44-625G

antibody from Invitrogen Antibodies

Targeting: PTK2 FADK, FAK, FAK1, PPP1R71

Provider product page for 44-625G

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Antibody data

Antibody Data
Antigen structure
References [53]
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Validations
Western blot [2]
Immunocytochemistry [1]
Other assay [42]

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Product number: 44-625G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-FAK (Tyr397) Monoclonal Antibody (141-9)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This antibody will cross-react with the corresponding autophosphorylation site on Proline-rich/Ca2+- activated tyrosine kinase (Pyk2), [pY402]. FAK [pY397] polyclonal antibody (Cat. No. 44-624G) does not cross-react with Pyk2.
Antibody clone number: 141-9
Concentration: 0.5 mg/mL

PTK2 protein structure - 44-625G shown in red.

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Experimental details: Figure 1 Combined proteomic and phosphoproteomic analysis of isolated adhesion complexes. ( a ) Schematic workflow for the isolation and proteomic/phosphoproteomic analysis of adhesion complexes. Cells were allowed to spread on FN or, as a control, Tf and complexes were isolated by a combination of crosslinking, cell lysis and a high-pressure wash to remove cell bodies. Collected complexes were analysed using either a proteomic or phosphoproteomic workflow, after which the FN-specific proteins and phosphoproteins were identified by performing a subtractive comparison with controls. ( b ) Immunoblot analysis of complexes isolated from cells spread on FN and Tf, as well as the WCLs of cells spread on FN. M, MW markers (kDa; values displayed to the left of each blot). Dashed lines indicate where images have been cropped for display purposes. ( c ) A Venn diagram showing the overlap between the FN-specific proteins (left circle) and phosphoproteins (right circle) identified by proteomic and phosphoproteomic analyses of isolated complexes, respectively. In addition to the total number of proteins (black text), the number of adhesome proteins identified in each data set is also displayed (red text). To the right of the panel, all 19 adhesome components identified exclusively by the phosphoproteomic analysis are displayed. Proteins in bold text were not identified by any other proteomic analyses of isolated FN-induced adhesion complexes.

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Experimental details: Figure 4. FAK E1015A expression inhibits talin binding and recruitment to nascent adhesions. (A) Cell lysates were prepared from FAK -/- MEFs stably re-expressing GFP, GFP-FAK WT, or GFP-FAK E1015A after replating on FN for 15 min. Anti-GFP immunoprecipitates were analyzed for endogenous talin and paxillin association. Lysates were also analyzed for talin, GFP-FAK, paxillin, and actin expression. (B) GFP-FAK WT and GFP-FAK E1015A MEFs were plated on FN for 15 min and analyzed for GPP-FAK (green) and paxillin (red) staining ( Fig. S3 A ). (C and D) In parallel, cells were analyzed for GPP-FAK (green) and talin (red) staining. Co-localization analyses and box and whisker plots are described in the legend for Fig. 1 . Representative images of FN-replated GFP-FAK WT and GFP-FAK E1015A MEFs are shown. The merged images show colocalization (yellow). Inset, enlarged area of peripheral adhesion staining (circled). Bars, 10 um. (E) FAK -/- MEFs expressing GFP, GFP-FAK WT, or GFP-FAK E1015A were serum starved, suspended (S), and FN replated for the indicated times. Anti-pY397 FAK immunoblotting shows FN-stimulated FAK activation with respect to total FAK and actin levels. Molecular mass is indicated next to the gel blots in kilodaltons. (F and G) Adhesion number (F) and size (G) were determined in the indicated cells at 60 min on FN by anti-paxillin staining in at least 10 cells per experimental group. Box and whisker plots are described in the legend to Fig. 1 (**, P < 0.01; *, P < 0.

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Experimental details: Figure 1. TNF-alpha-induced VCAM-1 expression within heart-associated ECs in vivo is dependent on FAK activity. PBS or TNF-alpha (0.02 mg/kg) was tail vein injected into mice, and, after 6 h, tissues were analyzed by immunoblotting or staining. Where indicated, FAK-I (100 mg/kg, PND-1186) was administered 3 h before starting experiments. (A) Immunoblotting of heart lysates shows increased VCAM-1 expression and FAK Y397 or FAK Y576 phosphorylation upon TNF-alpha stimulation in vivo. FAK-I addition prevents VCAM-1 production and FAK tyrosine phosphorylation but no change in FAK expression. Internal loading controls for each gel are shown by anti-actin immunoblotting. (B) Heart-associated VCAM-1 or FAK activation (pY397) was determined by immunoblotting (see Fig. S1 ) and expressed as a ratio to actin or total FAK, as determined by densitometry, respectively. Values are means (+-SD) from six mice, representing two independent experiments (***, P < 0.001). (C) In vivo signaling assays were performed as in A, and heart sections were analyzed by combined staining for activated FAK (pY576) and ECs (CD31). Bar, 20 um. (D) Mean correlation of pixel intensities from anti-pY576 FAK and anti-CD31 staining of heart sections, as shown in C. (E) Visualization of EC-associated VCAM-1 expression. Heart sections were analyzed by combined staining for VCAM-1 and ECs (CD31). A merged image is shown. Bar, 20 um. (F) Mean correlation of pixel intensities from anti-VCAM-1 and anti-CD31 staining of

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Experimental details: Figure 2. FAK activity controls developmental VCAM-1 expression. (A) FAK-KD embryos at E9.5 exhibit an enlarged and unfused allantois (arrow) compared with normal FAK-WT embryos where chorioallantoic fusion has occurred. (B) Treatment of pregnant female mice with FAK-I (100 mg/kg, PND-1186 from E7.5 to E9.5) but not vehicle (water) results in lethality of embryos (E9.5), exhibiting defective allantois formation (arrow). (C) Sagittal sections of E9.5 WT and FAK-KD embryos. FAK-KD embryos do not form somites (S) and display gross defects in head, heart (H), and allantois (Al). The arrow indicates the chorion (Ch) membrane. G, gut; N, neural cell. (D) Whole-mount anti-VCAM-1 staining of embryos at E9.5 reveals no VCAM-1 expression in FAK-KD compared with FAK-WT embryos. HD, head. (E) Lack of VCAM-1 staining in FAK-I (100 mg/kg, PND-1186 from E7.5 to E9.5) compared with vehicle-treated embryos at E9.5. (A-E) Bars, 100 um. (F-H) VCAM-1 protein expression and FAK activation are inhibited within FAK-KD and FAK-I-treated embryos. Immunoblotting of E9.5 embryo lysates reveals VCAM-1 levels (left) and FAK phosphorylation (pY397; right) with actin and total FAK as loading controls. Results from MEFs (G) or ECs (H) show that TNF-alpha (10 ng/ml, 16 h) induces VCAM-1 expression in FAK-WT but not FAK-KD cells, as determined by immunoblotting with pY397 FAK, total FAK, and actin levels, shown as controls.

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Experimental details: Figure 4. TNF-alpha-induced MAPK but not NF-kappaB activation is dependent on FAK activity in vitro and in vivo. (A and B) FAK-WT or FAK-KD MEFs (A) and HUVECs pretreated with DMSO or FAK-I (1 uM PF271; B) were stimulated with 10 ng/ml TNF-alpha for the indicated times, and lysates were prepared for immunoblotting. Blots for activated FAK (pY397), total FAK, activated NF-kappaB (pS536), activated JNK (p-JNK and pT183/pY185), activated ERK (pERK and pT202/pY204), IkappaBalpha, and actin are shown. Internal loading controls for each gel are shown by anti-actin, anti-GAPDH, anti-talin, or reprobing membranes with antibodies to total NF-kappaB, JNK2, or total ERK1/2 immunoblotting. (C) PBS or TNF-alpha (0.02 mg/kg) was tail vein injected into mice, and, after 5 min (FAK and ERK activation) or 3 h (NF-kappaB activation), lung tissue was analyzed by immunoblotting or EMSA. Where indicated, FAK-I (100 mg/kg, PND-1186) was administered 3 h before starting experiments. Values, measured by densitometry, are means (+-SD) from six mice, representing two independent experiments. ***, P < 0.001. (D and E) MEFs were replated onto FN dishes or held in suspension (Sus) for 1 h before TNF-alpha (10 ng/ml) addition for 15 min (D) or 6 h (E) before protein cell lysis. Blots for activated ERK (pERK and pT202/pY204), activated FAK (pY397), total FAK, and actin are shown. Anti-GAPDH blotting is shown as loading controls.

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Experimental details: Figure 5. FAK inhibition decreases GATA4 levels needed for TNF-alpha-induced VCAM-1 expression. (A) Steady-state GATA4 and GATA6 levels in FAK-WT and FAK-KD MEFs, as determined by immunoblotting with actin as a control. (B) FAK-WT MEFs treated with DMSO or FAK-I (1 uM PF271, 6 h) and lysates blotted for GATA4 or GATA6. Anti-GAPDH blotting is shown as loading controls. (C) GATA4 mRNA levels to GAPDH were determined by Q-PCR (+-SD; n = 3) in experiments, as described in B. (D) Rescue of TNF-alpha-induced VCAM-1 expression in FAK-KD MEFs by combined FAK-WT and GATA4 expression. Combinations of GFP-FAK and GATA4 were transfected into FAK-KD MEFs. After 24 h, cells were stimulated with 10 ng/ml TNF-alpha, as indicated, and FAK, VCAM-1, and GATA4 immunoblotting was performed at 40 h. (E) FAK-WT MEFs were transfected with Scr or GATA4 (G4) siRNA and, after 48 h, stimulated with TNF-alpha (10 ng/ml, 6 h), and immunoblotting was performed for VCAM-1 and GATA4. Anti-actin and anti-GAPDH blotting are shown as loading controls. (F) Densitometry analyses of VCAM-1 protein levels relative to actin, as described in E. (+-SD; n = 2; ***, P < 0.001). (G and H) MEFs were transfected with Scr or GATA4 siRNA and stimulated with TNF-alpha, as described in E. GATA4 (G) or VCAM-1 (H) mRNA levels to GAPDH were determined by Q-PCR (+-SD; n = 3; ****, P < 0.0001). (I) PBS or TNF-alpha (0.02 mg/kg) was tail vein injected into mice, and, after 6 h, heart lysates were analyzed by immunoblotting for VCAM-

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Experimental details: Figure 7. FAK-enhanced GATA4 polyubiquitination is dependent on CHIP and FAK-FERM nuclear localization. (A) FAK-WT, Mdm2 -/- p53 -/- , and CHIP -/- MEFs were transfected with flag-tagged GATA4 and treated with MG132 (40 uM, 3 h) and FAK-I (1 uM PF271), as indicated. Flag tag immunoprecipitates (IPs) were evaluated by anti-ubiquitin (Ub) and GATA4 immunoblotting and show no ubiquitination of GATA4 in CHIP -/- MEFs. (B) CHIP -/- MEFs were transfected with HA-tagged CHIP and flag-tagged GATA4 and treated with MG132 (40 uM, 3 h) and FAK-I (1 uM PF271). Flag tag immunoprecipitates were evaluated by anti-ubiquitin and GATA4 immunoblotting and show rescue of GATA4 ubiquitination by CHIP reexpression. Lysates show HA-CHIP and actin expression. (C) 293T cells were cotransfected with GFP-tagged FAK, FAK-FERM, and FAK-CT with HA-CHIP. Coimmunoprecipitation analyses with antibodies to GFP reveal FAK-FERM and CHIP association by immunoblotting. Lysates show equal levels of GFP-FAK and HA-CHIP expression. (D) 293T cells were transfected with GFP-tagged FAK-FERM WT or FAK-FERM R177A/R178A, flag-tagged GATA4, and His-tagged ubiquitin and denatured lysates (8 M urea) purified by nickel agarose affinity binding. GFP-FERM and Flag-GATA4 (with actin and GAPDH as loading controls; left), total eluted ubiquitinated proteins (middle), and mono-, di-, and polyubiquitinated GATA4 (right), as determined by immunoblotting, are shown. (E) FAK-WT MEFs pretreated with DMSO or FAK-I (1 uM PF271, 30 min) we

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Experimental details: Figure 3. FAK activity is essential for proinflammatory cytokine-mediated VCAM-1 expression. (A) TNF-alpha-induced (10 ng/ml, 16 h) VCAM-1 is prevented by FAK-I (PF271) treatment of HUVECs in a dose-dependent (0.1-2.5 uM FAK-I) manner. VCAM-1, pY397 FAK, total FAK, and actin levels were determined by immunoblotting. (B) TNF-alpha-induced VCAM-1 promoter activity is blocked by FAK inhibition. HUVECs were transfected with pGL3 promoterless luciferase (control) or pGL3 fused with the human VCAM-1 promoter, stimulated with TNF-alpha, and treated with FAK-I (1 uM PF271), as indicated, and relative luciferase activity (arbitrary units [a.u.]) is shown. Values are means (+-SD) from three independent experiments. (C) FAK knockdown prevents VCAM-1 expression. HUVECs were transduced with lentiviral Scr or anti-FAK short hairpin RNA (shRNA) and stimulated with TNF-alpha. FAK and VCAM-1 and actin expression levels were determined by immunoblotting, and densitometry values of VCAM-1 relative to actin are means (+-SD; n = 3; ***, P < 0.001). (D) VCAM-1 mRNA levels to GAPDH were determined by Q-PCR (+-SD; n = 3; ***, P < 0.001) in experiments, as described in C. (E) FAK activity is required for IL-1beta-induced VCAM-1 expression. HUVECs were stimulated with 20 ng/ml IL-1beta and 10 ng/ml TNF-alpha, and FAK-I (1 uM PF271) was added as indicated. After 16 h, VCAM-1, pY397 FAK, and actin levels were evaluated by immunoblotting.

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Experimental details: Figure 4 The effect of miR-125a-3p on hallmark genes in prostate cancer PC3 cells, transfected with miR-125a-3p or with empty vector (control), were cultured for 48 hours. Cells were lysed and, (A) their Fyn mRNA expression was analyzed by Real Time PCR. Proteins were analyzed by WB with specific antibodies against: (B) Fyn, (C) phospho-FAK (p-FAK) and (D) phospho-paxillin (p-paxillin), as well as against their loading control proteins (actin, general FAK and general paxillin, respectively). The experiment was repeated 3 times. Intensity of bands was analyzed using the image J software and the ratio between each protein and its control was plotted. The bars are mean+SD. (*) - Significantly different from control value (p

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Experimental details: Figure 5 Focal adhesion association of SRC controls SRC-mediated substrate phosphorylation, focal adhesion formation and cell migration. ( a ) Cell lysates from U2OS cells expressing GFP, SRC-GFP, SRCDeltaSH2-GFP, SRCY527F-GFP or SRCY527FDeltaSH2-GFP were analyzed by Western blotting for pY577-FAK, pY397-FAK, pY118-paxillin, pY31-paxillin, paxillin, GAPDH and GFP. ( b ) Cell lysates from SYF cells expressing GFP, SRC-GFP, SRCDeltaSH2-GFP, SRCY527F-GFP or SRCY527FDeltaSH2-GFP were analyzed by Western blotting for pY577-FAK, pY397-FAK, pY118-paxillin, pY31-paxillin, paxillin, GAPDH and GFP. ( c ) U2OS cells expressing GFP, SRCY527F-GFP or SRCY527FDeltaSH2-GFP (blue) were immunostained to detect F-actin (phalloidin; red) and paxillin (green), and imaged by epi-fluorescence and TIRFM, respectively. Scale bar, 10 mum. ( d ) The number of segmented paxillin-marked FAs within U2OS cells, as described in ( c ). Data are means +- s.e.m. (GFP: n = 11 cells; SRCY527F-GFP: n = 13 cells; SRCY527FDeltaSH2-GFP: n = 12 cells). ** p < 0.01; *** p < 0.001; NS, no significance. ( e ) Size distribution of segmented paxillin-marked FAs of U2OS cells, as described in ( c ). Data are means +- s.e.m. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, no significance. ( f ) The migratory behavior of U2OS cells expressing GFP, SRCY527F-GFP or SRCY527FDeltaSH2-GFP for 6 hrs. Cells were plated for 16 hrs, and then monitored for 6 hrs. (right) These images delineate the trajectory of the GFP-marked cells over a

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Experimental details: Figure 1. Inhibition of FAK activity by FAK [i] in human fibroblasts. (A) Immunoblotting of FAK phosphorylation sites FAK Y397 and FAK Y576 and total FAK in total cell lysates. HFF cells spread on FN for 1 h were treated with DMSO or the FAK inhibitor AZ13256675 (FAK [i]) for 1 h using half-log dilutions. Cells kept in suspension for 30 min (Susp) were used to detect basal FAK activity. Untreated cells spread on FN for 2 h were used to detect maximal FAK activity. FAK Y397 and FAK Y576 were used to assess FAK catalytic activity. Molecular mass values (kD) are displayed. (B and C) Quantification of immunoblotted membranes in A. FAK phosphorylation values FAK Y397 (B) and FAK Y576 (C) were normalized to total FAK (mean +- SEM, n = 3). (D) Dose-response curve using FAK Y397 as a readout for FAK activity to determine percentage inhibition relative to cells treated with DMSO. Gray lines and shading show values for suspension and untreated FN conditions (not used to calculate the trendline, mean +- SEM, n = 3). The 50% FAK Y397 inhibitory concentration (IC 50 ) was calculated as 0.11 uM FAK [i] using the formula y = 12.23ln( x ) + 76.99 ( y, percentage inhibition; x, FAK [i] concentration). (E) HFFs spread on FN for 1 h were treated with DMSO, 0.1 uM FAK [i], or 3 uM FAK [i] for 1 h. IACs were visualized by staining for FAK Y397 (green) and vinculin (red) by immunofluorescence. The actin cytoskeleton was visualized by staining with fluorophore-conjugated phalloidin. Bars: (main) 20

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Experimental details: Figure 5 The upregulation of CXCR4 by KLF8 leads to a feed-forward activation of FAK upstream of KLF8 ( A ) Overexpression of KLF8 causes CXCL12/CXCR4-dependent activation of FAK. Uninduced (U) and induced (I) 10A-iK8 cells were serum-starved for 24 hours followed by 3-hour treatment with AMD3100 (35 ng/ml) or mock treatment prior to CXCL12 (100 ng/ml) stimulation. Whole cell lysates were prepared for western blotting for FAK phosphorylation at Y397 (pFAK) and total FAK. ( B ) Overexpression of KLF8 induces CXCL12/CXCR4-dependent expression of endogenous KLF8. The 10A-iK8 cells were grown and treated similarly as in A except for inclusion of PF223, a FAK-specific inhibitor. Total RNA was prepared for qRT-PCR (top panel) and semi-quantitative RT-PCT (bottom panel). ( C ) Hypothetic model for the feed-forward signaling loop of KLF8 to CXCR4/CXCL12 to pFAK and back to KLF8.

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Experimental details: Fig. 4 Angpt1 deficiency enhances initial stages of distant metastasis. a Quantification of B16F10 cells in the lungs 24 h after tail vein injection in WT ( n = 6) and Angpt1 Delta/Delta mice ( n = 6). b Quantification of B16F10 cells in the lungs 4 h after tail vein injection in WT and Angpt1 Delta/Delta mice ( n = 4-5). c Representative fluorescent image of lung from WT and Angpt1 Delta/Delta mice 4 h after tail vein injection of GFP positive B16F10 cells. d Leakage experiments with cadaverin in WT and Angpt1 Delta/Delta mice, with and without tail vein injection of B16F10 cells. e Measurement of cross-sectional area of capillaries from micrographs of lungs from WT ( n = 4) and Angpt1 Delta/Delta ( n = 4) mice. f Quantification of microspheres in lungs 4 h after tail vein injection in WT ( n = 5) and Angpt1 Delta/Delta ( n = 5) mice** p < 0.01, *** p < 0.001. g Western blotting for FAK phosphorylation at Tyr397 in WT and Angpt1 Delta/Delta mice, with and without tail vein injection of B16F10 cells. h Quantification of B16F10 cell attachment to HUVECs with or without pre-treatment with Angpt1

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Experimental details: Figure 4 Validation of adhesion complex-localized phosphorylation sites. ( a ) Immunoblot analyses of the protein complexes isolated from cells spread on FN or Tf for 120 min. Samples were probed using phosphospecific antibodies targeting: paxillin pTyr118; FAK pTyr397, pTyr576 and pTyr407; MYPT1 pThr696; cortactin pTyr421; SHP2 pTyr580; and PKCdelta pTyr313. ( b ) Immunofluorescence images of cells spread on FN for 120 min. Cells were stained using the phosphospecific antibodies FAK pTyr407, cortactin pTyr421 and PKCdelta pTyr313 (all green). In each case, cells were also stained using an antibody targeting the focal adhesion marker paxillin (red). Scale bar, 20 mum. ( c ) Immunoblot analyses of the WCLs of cells spread on FN or Tf for 120 min. Samples were probed using the same antibodies as used to probe isolated complexes in a . Dashed lines in a , c indicate where the images have been cropped for display purposes. White arrows to left of blots highlight the expected MWs of the corresponding proteins. Black arrows to the left of blots highlight a putative high MW isoform of PKCdelta.

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Experimental details: Figure 4 ICAM2, Src, and annexin II increase during CdCl 2 -induced testis damage. Immunoblotting experiments showing changes in the levels of several proteins (A). Proteins whose levels increased following CdCl 2 treatment are labeled as bold. Actin was used as an internal control. Histograms (B) summarizing immunoblotting results. Each data point was normalized against its corresponding actin data point and then against '0 h', which was arbitrarily set at 1. Each bar represents mean+- s.d. of n =3-6 rats. * P

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Experimental details: Figure 3 FAK PRR2 and PRR3 regions are direct binding sites for cortactin. (A) Cortactin SH3 domain binds FAK as determined by GST or GST-cortactin SH3 domain pulldown assays using in vitro translated FAK protein. FAK immunoblotting shows binding and 5% of input. (B) Schematic of FAK containing an N-terminal FERM domain, three PRR (PRR1-PRR3) sites, a central kinase domain, and a C-terminal FA targeting region. Point-mutations are indicated that disrupt PRR2 (A712/713) and PRR3 (A876/877). The indicated FAK regions (below) were used as bait or prey in direct binding assays as either GFP, C-terminal TAP, or GST fusion proteins. (C) FAK C-terminal domain binds cortactin. GFP fusions of FAK 1-402, FAK 396-686, and FAK 411-686 with a C-terminal TAP tag or non-tagged FAK 686-1052 were in vitro translated in the presence of biotin-lysine and used in a direct binding assay with GST or GST-cortactin attached to beads. Streptavidin-HRP analyses show the amount of FAK bound or 5% of prey material used in the binding assay. (D) FAK PRR3 region binds cortactin. In vitro translated full-length cortactin was incubated with GST-FAK (853-946) or GST-FAK (947-1052) in a direct binding assay. Streptavidin-HRP analyses show the amount of cortactin bound or 5% of prey material used in the binding assay. (E) FAK PRR2 and PRR3 are individually required for cortactin binding. GFP-fusions of FAK WT, A713/713, or A876/877 were in vitro translated and incubated with GST-cortactin in a direct binding a

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Experimental details: Figure 5 Regulation of FAK and cortactin binding by FN adhesion. (A) Lysates were made from MEFs held in suspension for 30 min or FN re-plated (30 and 60 min) and were analyzed by cortactin or FAK antibody immunoprecipitation (IP) followed by immunoblotting for cortactin and FAK. (B) Mutations disrupting FAK activity or within PRR2 either stabilize or prevent cortactin association with FAK by co-IP analyses, respectively. Lysates were made from the indicated GFP-FAK reconstituted MEFs held in suspension for 30 min or FN plated (30 and 60 min) and were analyzed by anti-GFP IPs followed by anti-cortactin, anti-FAK Y397 phosphorylation (pY397), and anti-GFP immunoblotting to determine the level of GFP-FAK.

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Experimental details: Figure 7 FAK tyrosine phosphorylation of cortactin during FN replating of MEFs. (A) Lysates were made from the indicated GFP-FAK reconstituted MEFs held in suspension for 30 min or FN plated (30 and 60 min) and were analyzed by anti-cortactin IPs followed by anti-phosphotyrosine (pY) and anti-cortactin immunoblotting. (B) In vitro kinase assays using recombinant GST-Cortactin incubated in the presence (lanes 1, 2 and 4) or absence (lane 3) of recombinant FAK kinase, or presence (lanes 1,3 and 4) and absence (lane 2) of ATP. Proteins were analyzed by anti-phosphotyrosine (pY) immunoblotting and by Coomassie staining.

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Experimental details: Figure 5 Invasive characteristics at the edge of the flat disc-like colonies. A ) Western blots of E-cadherin and vimentin. HCT-116 cells were grown in 3-D matrigel with E or RNEW media for 6 days. Cells were then lysed and subjected to Western blot analysis to determine the levels of E-cadherin and vimentin as described in Materials and Methods. GAPDH was used as a loading control. Densitometry shown below each protein. B ) Confocal images of round or flat colonies of HCT-116 cells grown in 3-D with E or RNEW media for 6 days. a, c, and e: round colonies; b, d, f, and g: flat colonies; a-b: E-cadherin merged with DNA; c-d: beta-catenin merged with DNA, and e-g: actin merged with DNA, g: zoom of image f. C ) Western blots of total and phospho-FAK. HCT-116 cells were grown in 3-D matrigel for 6 days with E or RNEW media. Cells were then lysed and subjected to Western blot analysis to determine the levels of total and phospho-FAK as described in Materials and Methods. GAPDH was used as a loading control. D ) Quantitation and images of Ki67 positive cells. HCT-116 cells were grown in 2-D or 3-D conditions with E or RNEW media for 6 days, stained for Ki67 and DNA and the percentage of Ki67 positive cells was assessed by immunofluorescence. Results are expressed as mean percentage of Ki67 positive cells, +/- SEM, n=3. Fluorescent images of HCT-116 cells grown in 3-D with (a) E or (b) RNEW media for 6 days. Cells were stained for DNA (blue) and Ki67 (green).

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Experimental details: Figure 3 Effects of anti-beta1-integrin-Ab blocking treatment on FAK protein expression and phosphorylation as well as the expression of gelatinases (MMP-2 and MMP-9) in KG1a and K562 leukemic cell lines. Protein expression levels of FAK, pFAK at sites (Y397, Y925 and Y576/577), MMP-2 and MMP-9 were determined using western blot analysis in (A) KG1a and (B) K562 cells treated with doxycycline (1 ug/ml) or anti-beta1-integrin-Ab (100 ng/ml). GAPDH was used as the loading control. Ab, antibodies; FAK, focal adhesion kinase; MMP, matrix metalloproteinase; Y, tyrosine; Con, control; p, phosphorylated.

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Experimental details: Supplementary Figure 7

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Experimental details: Figure 3. Effects of the duration and timing of FAK inhibition on cell adhesion. (A) Immunofluorescence staining of HFF cells spread on FN for 1 h and treated with DMSO or 3 uM FAK [i] for 1, 2, 3, or 4 h. IACs were visualized by staining for FAK Y397 (green) and vinculin (red). Bars: (main) 20 um; (ROI) 5 um. Additional images of paxillin-positive areas and the actin cytoskeleton are shown in Fig. S2 A. (B) Quantification of images in A and Fig. S2 A . Quantification of the cell area covered by FAK Y397 -, vinculin-, and paxillin-positive areas, and the total cell area (mean +- SEM; n = 10 cells, FAK Y397 , vinculin, and paxillin; n = 20 cells, cell area). (C) Immunofluorescence staining of HFF cells treated with FAK [i] added to suspension or prespread cells. To examine effects on cell spreading and IAC formation, DMSO or FAK [i] was added to HFF cells kept in suspension and cells were plated onto FN-coated plates (Susp). To examine effects on IAC maturation, cells kept in suspension were plated onto FN-coated plates for 1 h and treated with DMSO or FAK [i] (Adh). In both cases, cells were fixed after 2 or 16 h total spreading times. IACs were visualized by staining for FAK Y397 (green) and vinculin (red). Bars: (main) 20 um; (ROI) 5 um. Additional images of paxillin-positive areas and the actin cytoskeleton are shown in Fig. S2 B. (D) Quantification of images in C and Fig. S2 B. Quantification of the cell area covered by FAK Y397 -, vinculin- and paxillin-positive areas, a

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Experimental details: Figure 6. Effects of FAK, Src and combined FAK and Src inhibition on IAC proteins and phosphorylation . (A-I) HFF cells spread on FN for 1 h were treated with DMSO, FAK [i], Src [i], or combined FAK [i] + Src [i] for 1 h. IACs were visualized by staining for pY (green) and alpha5 integrin (red; A-C), FAK Y397 (green) and vinculin (red; D-F) or paxillin Y118 (green) and paxillin (red; G-I). Bars: (main) 20 um; (ROI) 5 um. Graphs show quantification of the cell area covered by positive staining of the indicated protein (mean +- SEM, n = 10 cells). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; Kruskal-Wallis test with Dunn's post hoc correction (in C, F, and I, comparisons with cells treated with DMSO are shown). Representative images are shown.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Effects of FAK, Src, and combined FAK and Src inhibition on IAC composition. (A and B) HFF cells spread on FN for 1 h were treated with DMSO, FAK [i], Src [i], or combined FAK [i] + Src [i] for 1 h. IACs were isolated using the workflow in Fig. 2 A and were analyzed by SDS-PAGE (A) and immunoblotting (B). Representative total protein intensity values for each lane are indicated. Graph shows intensity values normalized to the DMSO condition (mean +- SEM, n = 3). Cell lysates from cells spread on FN (TCL) were used as a positive control. Molecular mass values (kD) are displayed. (C) For quantification of immunoblotted membranes in B, the band intensity values for each protein are shown relative to the DMSO condition (mean +- SEM, n = 3). Quantification was not performed for Tf receptor or BAK. (D) Total cell lysates collected from HFF cells spread on FN for 1 h and treated with DMSO or inhibitors for 1 h were analyzed by immunoblotting. Molecular mass values (kD) are displayed. (E) For quantification of immunoblotted membranes in D, phosphorylation values normalized to the corresponding total protein values are shown relative to the DMSO condition (mean +- SEM; n = 4, FAK Y397 , FAK Y576 , and paxillin Y118 ; n = 3, p130Cas Y249 ). t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Representative images are shown.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 F806 inhibits beta1 integrin activation in ESCC cells A. Cell lysates were immunoprecipitated (IP) with anti-active beta1 integrin antibody followed by probing with anti-total beta1 integrin antibody. Total cell lysates were immunoblotted for anti-total beta1 integrin, alpha5-integrin, beta4-integrin and beta-actin. B. Paraffin-embedded tumor tissues from xenograft models were immunohistochemistry staining for anti-active beta1 integrin. Original magnification, 400x; Scale bar, 50 mum. C. Total cell lysates were immunoblotted for anti-p-FAK, FAK, p-AKT, AKT, p-ERK, ERK and beta-actin. D. KYSE 510 cells were pre-incubated with anti-beta1 integrin or control IgG for 30 min and then seeded into FN-coated plates in the presence of F806 for 24 hr. The attached cells were recorded by MTT assay in each group. E. After si-NC or si-beta1 integrin transfection for 48 hr, KYSE510 cells were kept in FN-coated plates in the presence of F806 for 24 hr. The attached cells were recorded by MTT assay in each group. * P < 0.05 vs . control group; mean +- SD, n = 3-6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 FAK-paxillin interaction conditions FAK localization to FA. ( A ) Cell extracts from A375 melanoma cells transiently co-transfected with siFAK and wild-type FAK-GFP or FAK-I936/I998-GFP were analyzed by immunoprecipitation (IP) using anti-FAK Ab and blotted for paxillin and FAK. The expression level of proteins in the corresponding cell lysate is shown. ( B ) Representative Western blot showing A375 melanoma cells transfected as described above and blotted for P-Y397 FAK, FAK total, eGFP and actin. ( C ) Cells transiently co-transfected with siFAK and wild-type FAK-GFP or FAK-I936/I998-GFP were fixed and labelled for paxillin (purple), actin (red) and P-Y397 FAK (cyan). Scale bar: 10 um. ( D ) Histograms represent the mean normalized to control +- SD of FAK localization in melanoma cells. Signals were measured by reporting the P-FAK signal in FA over that in the cytoplasm from 3 independent experiments ***, p < 0.001; unpaired t -test compared to control condition.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4: Depletion of ILK decreases the activation and assembly of focal adhesion proteins. Immunoblotting analysis for GAPDH and (A) total FAK, pY397-FAK (pFAK), (B) total paxillin (Pax), and pY118-paxillin (pPax) in shcntl and shILK cells cultured on soft or stiff substrata. Quantification shows mean +- SD for three independent experiments; immunoblotting replicates and statistical analysis are shown in Supplemental Figure S4. Immunofluorescence analysis for F-actin (green), nuclei (blue), and (C) FAK (magenta), (D) pFAK (magenta), (E) Pax (magenta), or (F) pPax (magenta) in shcntl and shILK cells cultured on plastic. Scale bars, 20 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7. A FAK signature reveals a graded response to stromal and epithelial FAKi priming before gemcitabine/Abraxane. ( A ) Schematic representation of PDAC PDX, PDCL, and PDX TMA establishment. ( B ) Representative images of TKCC05 and TKCC10 PDX TMA samples stained with total and pTyr 397 -FAK. Scale bars, 50 mum; n = 3 cores per PDX. ( C ) Quantification of pTyr 397 -FAK levels in PDCLs [mass spectrometry, top; data obtained from ()] and confirmation via Western blot (bottom), highlighting TKCC05 (orange; low active, high total FAK) and TKCC10 (purple; high active, low total FAK) cells. ( D to G ) Representative images (D and F) and quantification (E and G) (normalized cell number at >200-mum depth) of green fluorescent protein (GFP)-stained TKCC05 cells (D and E) and pan-cytokeratin-stained TKCC10 cells (F and G) invading into organotypic matrices primed with vehicle or FAKi (dotted line, 200-mum invasion depth). Scale bars, 50 mum. n = 3 biological repeats, 3 matrices per repeat, 3 FOVs per matrix. Results: means +- SEM. P values were determined using an ordinary one-way ANOVA with Tukey correction for multiple comparisons. Unless otherwise stated, all significance is compared to vehicle. ( H ) Timeline for TKCC05 and TKCC10 orthotopic studies. IVIS, In Vivo Imaging System. ( I to L ) Kaplan-Meier analysis of survival (I and K) and time to metastasis (J and L) in mice with TKCC05 (I and J) or TKCC10 (K and L) orthotopic tumors treated with vehicle/saline (TKCC05, n = 10;

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 PDAC genotype tunes epithelial tension to regulate fibrosis ( a ) SHG images from 20 week KC, KPC or KTC transgenic pancreatic tissues (top); Scale bar, 75 um. Force maps from AFM PDAC ECM (top insert). Immunofluorescence images of p-Mlc2, p-MyPT1 (insert), beta1-integrin and p-Ptk2, Yap1 and DAPI; Scale bar, 50 um. Tenascin C, Fibronectin (insert), Collagen type XII alpha1 and DAPI; Scale bar, 75 um. ( b ) Quantification of SHG fibril thickness and distribution around PDAC lesions. ( c ) Distribution of PDAC ECM stiffness measured by AFM. ( d ) Quantification of Tenascin C, Fibronectin and Collagen type XII alpha 1 images shown in (a). ( e ) Quantification of traction force KC, KPC and KTC cells on 2300 Pa polyacrylamide gels. ( f ) Quantification of mean collagen fiber diameter in three-dimensional collagen gels with KC, KPC or KTC cells or with KTC cells treated with vehicle or ROCK inhibitor Y27632 at 24 hours. ( g ) PR staining of tissue excised from nude mice 3 weeks after injection with KC, KPC, or KTC cells expressing either a control shRNA or an shRNA to Rock1 (top); Scale bar, 75 um. Immunofluorescence of p-Mlc2, p-MyPT1 (insert), Tenascin C, Yap1 and DAPI; Scale bar, 50 um. ( h ) Quantification of stiffness of tissue in (e). ( i ) Quantification of total levels of fibrillar collagen. For in vitro bar graphs, 3 technical replicates were performed and results are the mean +/- SEM of 3 independent experiments. For in vivo experiments, n = 5 mice per group. Su

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Tumor cell tension accelerates PDAC progression in mice ( a ) Cartoon of mouse manipulations used to study the impact of increasing pancreatic epithelial cell mechanosignaling using beta1-V737N on Kras-induced pancreatic malignancy. ( b ) Immunofluorescence images and quantification of pancreatic tissues from 3 month old KC and KC/beta1-V737N mice stained for p397-Ptk2 and p-Mlc2, Tenascin C, Yap1, p-Stat3; Scale bar, 50 um, CD68 and DAPI; Scale bar, 100 um. Polarized light images of PS staining; Scale bar, 75 um. Force maps of ECM stiffness. ( c ) Alcian blue H&E images of KC and KC/beta1-V737N tissue; Scale bar, 100um. ( d ) Quantification of histopathologic phenotypes present in KC and KC+beta1-V737N mice. ( e ) Polarized light images and quantification of PS staining of pancreatic tissues in nude mice injected with KTC tumor cells expressing either a control shRNA or a FAK shRNA; Scale bar, 75 um. Immunofluorescence images and quantification of Tenascin C (scale bar 75 um), Yap1 (75 um), p-Stat3 (scale bar, 25 um) and DAPI. ( f ) Quantification of average elastic modulus (Pa). For in vivo experiments, n = 5 mice per group. Subsequent statistical analysis was performed with unpaired two-sided student t-tests.. (* P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ""ns"" not significant).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Stat3 induces fibrosis and accelerates PDAC ( a ) Cartoon depicting the mouse crosses used for the activated Stat3C manipulations. ( b ) Immunofluorescence images and quantification of pancreatic tissues from 5 week old KC and KC/Stat3C mice stained for p-Stat3, scale bar, 75 um, p397-Ptk2 and p-Myl2, scale bar, 50 um, CD68, scale bar, 100 um, and DAPI. Polarized light images and quantification of PS staining; Scale bar, 75 um and H&E staining; Scale bar, 100 um. ( c ) Kaplan-Meier graph showing survival of KC and KC/Stat3C mice. ( d ) Quantification of ECM stiffness in (b). ( e ) Cartoon depicting the mouse crosses used for the Stat3 knock studies. ( f ) Immunofluorescence images and quantification of pancreatic tissues from 5 week old homozygous KTC (Control) and KTC+Stat3 -/- mice (KTC Stat3 -/- ) stained for p-Stat3, scale bar, 75 um, p397-Ptk2 and p-Myl2, scale bar, 50 um, Tenascin C, CD68, scale bar, 100 um, Yap1 scale bar, 50 um, and DAPI. Polarized light images and quantification of PS staining; Scale bar, 75 um, and H&E staining. Scale bar, 100 um. ( g ) Kaplan-Meier graph showing survival of KTC and KTC/Stat3 KO mice. ( h ) Quantification of ECM stiffness on tissue in (f). For in vivo experiments, n = 5 mice per group. Subsequent statistical analysis was performed with unpaired two-sided student t-tests. (* P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ""ns"" not significant).