44-722G

antibody from Invitrogen Antibodies

Targeting: PXN

Provider product page for 44-722G

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Antibody data

Antibody Data
Antigen structure
References [76]
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Validations
Western blot [3]
Other assay [38]

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Product number: 44-722G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-Paxillin (Tyr118) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: The antibody has been negatively preadsorbed using i) a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated paxillin, and ii) a generic tyrosine phosphorylated peptide to remove antibody that is reactive with phosphotyrosine, irrespective of the sequence. The final product is generated by affinity chromatography using a paxillin-derived peptide that is phosphorylated at tyrosine 118.

PXN protein structure - 44-722G shown in red.

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of COS-7 (Lane 1), COS-7 treated with 100 uM Pervanadate for 1 hour (Lane 2), A431 (Lane 3), A431 treated with 100 uM Pervanadate for 1 hour (Lane 4), HeLa (Lane 5) and HeLa treated with 100 uM Pervanadate for 1 hour (Lane 6).The blots were probed with Anti-Phospho-Paxillin pTyr118 Rabbit Polyclonal Antibody (Product # 44-722G, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 66 kDa band corresponding to Paxillin pTyr118 was enhanced across treated cell lines. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Western Blot analysis using Rabbit anti Paxillin (pY118) Polyclonal Antibody (Product # 44-722G)

Supportive validation

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of COS-7 (Lane 1), COS-7 treated with 100 uM Pervanadate for 1 hour (Lane 2), A431 (Lane 3), A431 treated with 100 uM Pervanadate for 1 hour (Lane 4), HeLa (Lane 5) and HeLa treated with 100 uM Pervanadate for 1 hour (Lane 6).The blots were probed with Anti-Phospho-Paxillin pTyr118 Rabbit Polyclonal Antibody (Product # 44-722G, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 66 kDa band corresponding to Paxillin pTyr118 was enhanced across treated cell lines. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 7 Loss of SNAP29 affects the pattern of focal adhesion complexes. (A-L) Control (SV80 and F572T) and CEDNIK (F110T or F165T) fibroblasts were fixed and stained with anti-FAK (A-C), anti-p-FAK (D-F), anti-PAX (G-I) and anti-p-PAX antibodies (J-L). Nuclei were stained with Hoechst. Bar, 10 um. (M) Western blot analysis showing the expression of FAK, p-FAK, PAX and p-PAX in control (F572T) and CEDNIK (F165T, F110T) cells. Anti-ERK antibodies were used as a loading control. The blot is a representative of 5 independent experiments. (N) Bar graph illustrating the expression levels of the focal adhesion components: FAK, p-FAK, PAX and p-PAX. Densitometry was used to quantify the amount of each focal adhesion component as seen in Western blot, using the Image Densitometer 1Dprime. To normalize the results, the intensity of each band was divided by the intensity of ERK in the same lane. The value obtained for control cells (F572T) was considered as 100%. (O) Bar graph showing quantitation of the intensity of FAK, p-FAK, PAX and p-PAX staining, measured in 14-168 cells, using the ImageJ software. The value obtained for control cells (SV80) was considered as 100%. Expression pattern of FAK, phospho-FAK, paxillin and phospho-paxillin is different in CEDNIK cells in comparison to control cells. However, their total level is similar.

Supportive validation

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Experimental details: Figure 5--figure supplement 1. FAK phosphorylation in Tln Ctr , Tln Ko , Tln Ko+T1V , Kind Ctr , Kind Ko and Kind Ko+K2GFP cells. ( A ) Densitometric quantification of western blot signals of lysates from untreated, EGF- and Mn 2+ -treated Kind Ctr , Tln Ko and Kind Ko cells seeded either on FN or PLL and probed with anti-Tyr-397 phosphorylated FAK (pY397-FAK) antibodies (n=3 independent repeats; significances are calculated with respect to PLL adherent cells; error bars indicate standard error of the mean). ( B , C ) Western blotting of indicated signaling proteins in untreated, EGF- and Mn 2+ -treated Tln Ctr , Tln Ko and Tln Ko cells re-expressing Venus-tagged talin-1 (Tln Ko+T1V ) ( B ), and Kind Ctr , Kind Ko and Kind Ko cells re-expressing GFP-tagged kindlin-2 (Kind Ko+K2GFP ) ( C ) seeded either on FN or PLL. EGF, epidermal growth factor; FAK, focal adhesion kinase; K2GFP, green fluorescent protein-tagged kindlin-2; FN, fibronectin; GFP, green fluorescent protein; PLL, poly-L-lysine; T1V, Venus-tagged full length talin-1. DOI: http://dx.doi.org/

Supportive validation

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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 3. Symmetric material exchange between stationary focal adhesions and cytosol. ( A ) The symmetric and asymmetric models of material exchange between adhesion sites and cytosol. In symmetric exchange a component, A , exits from adhesion site in the same state it had upon entering to it. In asymmetric exchange A exits in a different, primed state A* and relaxes back to state A in the cytosol, thereby generating a spatial gradient of the primed state emanating from adhesion sites. ( B ) Formulation of the two models for the case in which priming (of A ) is based on interaction (with protein B ). Here, asymmetric exchange would generate a spatial gradient of AB complex concentration emanating from adhesion sites. ( C - F ) Discriminating between the two models by measuring the physical associations near (= 9) ( E ) or for the median score of each pair +- MAD ( F ). Data-points far from the equality diagonals (dashed red lines) would correspond to asymmetric exchanges, as illustrated in ( D ), while data-points along the diagonal indicate symmetric exchange. Histogram in ( D ) shows the distribution of the difference in association scores near and far from focal adhesions. ( G ) FLIM images color-coding the fraction, alpha, of

Supportive validation

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Experimental details: Figure 3--figure supplement 1. FAK, paxillin, and CAS are tyrosine-phosphorylated and interact with SH2 domain in focal adhesions. ( A ) Immunofluorescence images of REF52 stained for FAK pY407, paxillin pY118, and CAS pY165 using phospho-specific antibodies. Scale bars, 15 mum. ( B ) A REF52 cell co-expressing meGFP-dSH2 and TD-mKate2-paxillin. Note the localization of dSH2 in cell-matrix adhesion sites, as previously reported ( Kirchner et al., 2003 ). Scale bar, 10 mum. ( C ) Top and middle rows, FLIM images color-coding the fraction (alpha) of the donor- (mCitrine) tagged protein (FAK, paxillin, CAS, or ILK) that FRET to the acceptor- (mCherry) tagged dSH2 before and after acceptor photobleaching. ILK is used here as a negative control for tyrosine phosphorylation. Note that low levels of alpha are observed also for ILK in focal adhesions, plausibly due to density effects. However, FAK, paxillin, and CAS exhibit higher alpha values, suggesting FRET due to direct interaction. Scale bars, 10 mum. DOI: http://dx.doi.org/

Supportive validation

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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.

Supportive validation

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Experimental details: Figure 2 Focal adhesion kinase activity, but not SRC activity, positively regulates the abundance of SRC_SH2 domain within focal adhesions. ( a ) Total cell lysate from untreated U2OS cells (-) or cells treated with FAKi (focal adhesion kinase inhibitor; 50 muM, 1 h), PP2 (SRC inhibitor; 10 muM, 1 h) or SU6656 (SRC inhibitor; 5 muM, 1 h) were analyzed by Western blotting to detect pY118-paxillin, pY31-paxillin, SRC, FAK, paxillin and GAPDH. ( b ) U2OS cells transfected with pGFP-C1, pGFP-SRC(SH2) or pGFP-SHP2(NSH2) were untreated (-) or treated with FAKi or PP2, and immunostained to detect paxillin. TIRFM images of GFP, GFP-SRC(SH2) or GFP-SHP2(NSH2) (green) and paxillin (red). Bar, 10 mum. The 20 mum x 20 mum areas indicated in the upper images are magnified in the images below. Values indicate Pearson's correlation coefficients (PC) of images of paxillin-Cy5 and GFP, GFP-SRC(SH2) or GFP-SHP2(NSH2) in U2OS cells untreated (-) or treated with FAKi or PP2. Data are means +- s.e.m. (GFP: n = 3 cells (-), n = 4 cells (FAKi), n = 6 cells (PP2); GFP-SRC(SH2): n = 7 cells (-), n = 6 cells (FAKi), n = 7 cells (PP2); GFP-SHP2(NSH2): n = 5 cells (-), n = 5 cells (FAKi), n = 4 cells (PP2)). ** p < 0.01; NS, no significance. ( c ) Time-lapse TIRFM images of U2OS cells co-transfected with mApple-paxillin (red) and pGFP-C1, pGFP-SRC(SH2) or pGFP-SHP2(NSH2) (green) during FAKi or PP2 treatment. The time is shown in hour. Bar, 10 mum. ( d ) The ratio of the average density (intensity p

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Experimental details: Figure 8 Vinculin activation and nano-scale localization are spatiotemporally regulated (a) Top: mTurquoise (mTurq) and processed mTurquoise/NeonGreen FRET ratio images of an HFF cell expressing the vinculin FRET biosensor. Bottom: Zoom of outlined area. FA mask (grey lines) created from the mTurq image and superimposed onto FRET ratio image. (b) mTurquoise intensity and FRET ratio along the 5um long line in the zoom in (a). Dark shaded areas are cytosolic (cyto) regions adjacent to the FA (light shaded area) (c) Mean FRET ratio quantification of the distal (Dist, facing cell edge) 1/3 and proximal (Prox, facing cell center) 1/3 of FA linescans. (d, h) Representative iPALM renderings of HFFs expressing vinculin-N-tdEos (d) or talin-C-tdEos (h). Histograms of the Z-position of molecules within Dist and Prox thirds displayed next to the colourscale. (e, i) Mean of Z-median measurements from Dist or Prox thirds (f, j) Averaged Z-position frequency histograms of molecules. Solid line, mean frequency; Shaded region, bootstrapped 95% confidence intervals. (g, k) Mean fraction of molecules localized to the three FA layers. (l) Time-lapse TIRFM images of FAs during blebbistatin washout. HFFs expressing paxillin-mCherry (top) and vinculin-EGFP (bottom) treated with 50uM blebbistatin for 2 hours. Blebbistatin washout at t=0 min, Arrow: newly formed FA. Boxed areas: time points for iPALM analysis. Scale bar= 2mum. (m, n) Representative iPALM renderings of vinculin-N-tdEos at 1min (m) an

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Experimental details: Figure 5. The role of substrate adhesion in neuronal migration and force generation. (a) Pulling assay. Pulling the LP of GCs at the middle point. Broken lines show the location of soma and LP growth cone before and after pulling. Bars, 10 um. (b) Summary of results of the pulling experiments. Under migratory or stationary conditions, the number of GCs that showed the first detachment site at the soma or at the growth cone of LP after pulling is shown. (c) Immunostaining of markers of adhesion complexes (FAK, paxillin, vinculin). (d) FAK inhibition with Y11 (~50-200 uM) enhanced the strain energy of GCs ( n = 7). Broken lines show the normalized strain energy (1 unit) before drug application.

Supportive validation

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Experimental details: Figure 2 Automated image analysis of focal adhesion morphology allows quantitative assessment of different dynamic states of FAs. ( a ) Automated image analysis identifies individual focal adhesions based on pTyr118-paxillin or vinculin staining. ( b ) Image analysis and quantification of focal adhesion sizes in a set of representative images of the nocodazole assay. Focal adhesion size distribution of nocodazole-induced FA assembly and microtubule-induced FA disassembly were compared using a Kolmogorov-Smirnov (KS) test.

Supportive validation

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Experimental details: FIGURE 9: Enhanced paxillin phosphorylation permits invasion in cells with a basal phenotype. (a) Paxillin localization to the basal surface in MCF10A cells with and without TGFbeta treatment visualized with immunofluorescence staining and TIRF imaging of paxillin and phosphorylated paxillin, pPax[31]. Scale bar, 10 um (main image), 1 um (inset). (b) Paxillin expression in PyMT, Met1, and PyMT + TGFbeta visualized with immunofluorescence staining and confocal imaging of paxillin and phosphorylated paxillin pPax[31]. Scale bar, 10 um (main image), 1 um (inset). (c) Paxillin expression of PyMT cell spheroids visualized in soft and stiffened 3D collagen gel. Leader cells in stiffened gel have higher levels of phosphorylated paxillin indicated by pPax[31] immunostaining. Scale bar, 20 um (main image), 10 um (inset). (d) A 3D collagen invasion assay of multicellular spheroids composed of Met1 cells overexpressing either paxillin WT or paxillin Y31/118F in 2.5 mg/ml 3D collagen gels for 72 h. Scale bar, 100 um. (e) Quantification of invasion. Paxillin WT overexpression significantly increased invasion compared with control or paxillin Y31/118F overexpression, as determined by cross-sectional area of spheroid and protrusions and single-cell invasion (* p < 0.05; >20 spheroids). (f) A 3D collagen invasion assay of multicellular spheroids composed of metastatic Met1 cells treated with dimethyl sulfoxide or 20 nM dasatinib, a Src/Abl inhibitor, in 2.5 mg/ml 3D collagen gels for 72 h. D

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Experimental details: Supplementary Figure 7

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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

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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

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Experimental details: Figure 1 A nocodazole-based microtubule depletion assay to study focal adhesion dynamics in high-throughput fashion. ( a ) Treatment of MCF7 WT cells with nocodazole (10 muM, 4 h) depolymerized microtubules (MT), leading to activation of the actin cytoskeleton and focal adhesion assembly. Washout of nocodazole allows MT regrowth, resulting in focal adhesion disassembly. MCF7 WT cells were stained for vinculin and phosphorylated paxillin to visualize focal adhesions. ( b ) Schematic representation of screen setup. Transfection was performed in 96-well plates and controls were included in each plate. MCF7 WT cells were exposed as indicated, to induce FA assembly and disassembly. Plates were stained for vinculin and phosphorylated paxillin and imaged with automated confocal microscopy. ( c ) Schematic representation of cellular and FA organization in the nocodazole assay, with possible FA phenotypes caused by siRNA knockdown.

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Experimental details: 3 Figure S100A4 depletion inhibits the membrane localization and activation of vinculin and paxillin, and S100A4 depletion inhibits actin polymerization but does not affect myosin RLC phosphorylation A and B , in situ PLA fluorescence and PLA fluorescence merged with phase contrast from unstimulated (US) or ACh stimulated cells freshly dissociated from sham-treated or S100A4-depleted tissues. A , paxillin-vinculin complexes are distributed throughout the cytoplasm of unstimulated cells from both sham-treated and S100A4-depleted tissues. ACh stimulation resulted the localization of paxillin-vinculin complexes to the membrane of sham-treated cells (US, n = 13; ACh, n = 15, p = 0.0001), whereas paxillin-vinculin complexes remained distributed throughout the cytoplasm of S100A4-depleted cells (US, n = 12; ACh, n = 14, p = 0.0001). B , ACh stimulation resulted in a marked increase in the number of talin-vinculin complexes at the cell membrane in sham-treated cells (US, n = 21; ACh, n = 29, p = 0.0001). S100A4 depletion inhibited the ACh-induced increase of the interaction between talin and vinculin at the cell membrane (US, n = 26; ACh, n = 30, p = 0.0681). Few spots were observed in S100A4-depleted treated cells with or without stimulation. C , representative immunoblots from extracts of sham-treated muscle tissues and S100A4 shRNA-treated tissues stimulated with ACh or not stimulated (US). The increases in vinculin Tyr1065 phosphorylation and paxillin Tyr118 phosphorylation in r

Supportive validation

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Experimental details: 6 Figure Depletion of rhotekin inhibits NM myosin II polymerization and membrane recruitment and activation of vinculin and paxillin in response to ACh A , immunoblot of NM myosin II in cytoskeletal (P) and soluble (S) fractions from extracts of tracheal SM tissues treated with RTKN siRNA or untreated (Sham) and stimulated for 5 min with 10 -5 M ACh or unstimulated (US). RTKN depletion significantly inhibited the ACh-induced increase in the ratio of NM myosin II in the cytoskeletal (P) vs . soluble (S) fractions ( n = 6, p = 0.0001). B , in situ PLA fluorescence in freshly dissociated US or ACh-stimulated SM cells from sham-or siRNA-treated tracheal SM tissues. ACh stimulation of sham-treated tissues resulted in a significant increase in the number of NM myosin IIA and IIB complexes at the cell cortex ( n = 20 cells/group). RTKN depletion inhibited the ACh-induced increase in the interaction between NM myosin IIA and IIB ( n = 20 cells/group, P = 0.0001). C , RTKN depletion significantly inhibited the increase in NM myosin II Ser1943 phosphorylation in tracheal muscle tissues in response to 5 min stimulation with 10 -5 M ACh ( n = 11, P = 0.0001). D , in situ PLA fluorescence shows interactions between talin and vinculin in US or ACh-stimulated freshly dissociated SM cells from sham-treated or RTKN-depleted tissues. ACh stimulation resulted in a significant increase in the number of talin-vinculin complexes at the cell membrane (US cells, n = 19; ACh, n = 25 cells, p = 0.0001

Supportive validation

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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

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Experimental details: Figure 1 Cell migration and focal adhesion formation in MSCs compared to other primary mesodermal-derived adherent cells. ( A ) Average migration velocity of ABMSC compared to FBMSC, HUVEC and fibroblasts, measured by live cell phase contrast microscopy. Images were obtained every 30 minutes for a total of 12 hours at 37 degC, 5% CO 2 and migration was tracked using ImageJ. Median +- range. n = 4 independent experiments including 30 cells each. ns = non-significant, *p < 0.05, ***p < 0.001 (Kruskal-Wallis, multiple comparisons uncorrected Dunn's test). ( B ) Maximum absolute travel distance from point origin of ABMSC, FBMSC, HUVEC and fibroblasts within 12 hours. Median +- range. n = 4 independent experiments, including 30 cells each. ns = non-significant, **p < 0.01 (Kruskal-Wallis, multiple comparisons uncorrected Dunn's test). ( C ) Graph shows ABMSC migration velocity on varying concentrations of human-derived fibronectin (hFN) and bovine-derived fibronectin (bFN). Images were obtained every 30 minutes for a total of 12 hours at 37 degC, 5% CO 2 and migration was tracked using ImageJ. Median +- range. n = 4 independent experiments comprising cells from 4 different donors, analysis includes 15 cells per condition. Spearman correlation test. ( D ) Average migration velocity of ABMSCs cultured in either Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS) or with 5% Platelet lysate (PL) and cultured on 20 ug/ml hFN or bFN. n = 4 independent experiments c

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Experimental details: Figure 2 Presence of focal adhesions at endothelium from the umbilical cord. (A) Immunofluorescent staining for phospho-paxillin (green) and VE-cadherin (magenta) of HUVECs grown on glass substrate. ROI is indicated in the merge. Images are inverted for clarification. Lower panel shows zoom from ROI. Scale bar, 50 um. (B) Immunofluorescent staining for phospho-paxillin (green) and VE-cadherin (magenta) of HUVECs on an umbilical cord. ROI is indicated in the merge. Images are inverted for clarification. Lower panel shows zoom from ROI. Scale bar, 50 um (C) Immunofluorescent staining for phospho-paxillin (green), F-actin (magenta), VE-cadherin (cyan) and nuclei (blue) of BOEC seeded on umbilical cord, treated with TNF-alpha. ROI is indicated in the merge. Images are inverted for clarification. Lower panel shows zoom from ROI. Black arrowheads indicate focal adhesions. Scale bar, 50 um.

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Experimental details: Figure 5 Focal adhesions upon RhoJ silencing. (A) Immunofluorescent staining for phospho-paxillin (green), F-actin (magenta), VE-cadherin (Turquoise) and DNA (blue) on HUVECs stimulated for 20h with TNFalpha. ROI is indicated in the merge. Images are inverted for clarification. Upper panel are control cells (shCtrl), lower panel are cells with RhoJ depletion (shRhoJ). Scale bar, 20 um. (B) Quantification of the number of focal adhesions (FA) per field of view (FOV) for control (shCtrl) and RhoJ knockdown (shRhoJ) conditions. Superplot in which small dots are individual datapoints, large dots represent means from 5 independent experiments (five colors). Mean and standard deviation are indicated. Welch's T-test *p=0.000218. (C) Quantification of the size of focal adhesions (FA) for control (shCtrl) and RhoJ knockdown (shRhoJ) conditions. Superplot in which small dots are individual datapoints, large dots represent means from 5 independent experiments (five colors). Mean and standard deviation are indicated. Welch's T-test p=0.2712415, not significant (n.s.). (D) Quantification of the speed of basolateral neutrophils under control (shCtrl) and RhoJ knockdown (shRhoJ) endothelial cells. Violin plot in which small dots are individual datapoints. Mann-Whitney U-test: p

Supportive validation

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Experimental details: Figure 7 Focal adhesions upon Tiam1-C1199 expression. (A) Immunofluorescent staining for phospho-paxillin (green), F-actin (magenta), VE-cadherin (Turquoise) and HA (blue) on HUVECs stimulated for 20h with TNFalpha. Images are inverted for clarification. Upper panel are control cells (Ctrl HA), lower panel are cells expressing Tiam1-C1199 (Tiam1-C1199). Scale bar, 20 um. (B) Quantification of the area focal adhesions (FA) cover or control (Ctrl) and Tiam1-C1199-expressing cells (Tiam1-C1199). Superplot in which small dots are individual datapoints, large dots represent means from 3 independent experiments (three colors). Mean and standard deviation are indicated. Welch's T-test *p=0.02411431. (C) Quantification of the number of focal adhesions (FA) per field of view (FOV) for control (Ctrl) and Tiam1-C1199-expressing cells (Tiam1-C1199). Superplot in which small dots are individual datapoints, large dots represent means from 3 independent experiments (three colors). Mean and standard deviation are indicated. Welch's T-test *p=0.04694375. (D) Quantification of the size of focal adhesions (FA) in control (Ctrl) and Tiam1-C1199-expressing cells (Tiam1-C1199). Superplot in which small dots are individual datapoints, large dots represent means from 3 independent experiments (three colors). Mean and standard deviation are indicated. Welch's T-test *p=0.02819648 (E) Quantification of the percentage of TEM events in which a neutrophil migrates at a control-Tiam1-C1199 junction and mi

Supportive validation

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Experimental details: Figure EV3 Subcellular localization of EVL and postnatal angiogenesis in VASP -/- mice A EVL localizes to focal adhesions in endothelial cells. MLEC from wild-type mice were stained for phospho-paxilin (green) as a marker for focal adhesions, EVL (red) and actin (blue). White arrows indicate integrin-based focal adhesions at the tips of actin stress fibers. Representative images from three independent experiments are shown. Scale bar, 10 um. B, C Postnatal retinal angiogenesis in VASP -/- mice. (B) Isolectin B4-stained vasculature in whole mount retinas of wild-type (WT) and global VASP -/- mice on postnatal days 3 and 5 (P3, P5) assessed by confocal microscopy. Scale bars 200 um. (C) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates. Error bars represent SEM; no significant difference was observed between the two genotypes at P3 ( P > 0.999) or P5 ( P > 0.999) (one-way ANOVA with Bonferroni's multi-comparison test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 7. c-Src mediates paxillin phosphorylation and subsequent focal adhesion formation. (A) Endothelial cells were isolated from lungs of c-Src flox/flox and c-Src flox/flox ; Cdh5-CreERT2 mice, and protein expression was assessed by western blot. (B) A decrease in phospho-paxillin at Y118 was observed ( n >5 mice). (C) HUVECs transfected with siRNA for control or c-Src, or with overexpression of empty-mKate2 or c-Src-mKate2 cultured on fibronectin-coated coverslips were immunostained for p-paxillin-Y118 (pPaxY118), c-Src (for siRNA-treated cells), VE-cadherin and DAPI. c-Src-mKate2 is visualized with mKate2 signal. Single channel images of pPaxY118 are shown on the right; mKate2-positive cells are outlined with dashed lines. Scale bars: 20 um. (D-F) Focal adhesion number, density and size based on pPaxY118 staining was significantly increased upon c-Src-mKate2 overexpression ( n >30 cells in total from three independent experiments). (G,H) HUVECs transfected with siRNA for control or c-Src cultured on vitronectin-coated coverslips were stained for p-paxillin-Y118 (pPaxY118), VE-cadherin and DAPI. Single channel images of pPaxY118 are shown on the right. Scale bars: 20 um. (I-K) Focal adhesion number and density based on pPaxY118 staining was significantly decreased upon c-Src depletion, while the size was not significantly changed. n >15 cells in total from three independent experiments. (L-O) c-Src flox/flox ; mTmG and c-Src flox/flox ; mTmG; Cdh5-CreERT2 retinas were immu

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Combination therapy with gemcitabine and proglumide decreases metastases by preventing epithelial to mesenchymal transition (EMT). Quantitative PCR analysis for mRNA expression of transcription factors and proteins from PDAC tumors ( n = 4 per group) with relative gene expression (2 -DeltaDeltaCt +- SEM) for: ( A ) Vimentin , ( B ) Zeb1 , ( C ) Zeb2 , ( D ) SNAIL1 , ( E ) beta-CATENIN , and ( F ) TBFbetaR2 . Unpaired t-test was used for analysis (* p = 0.05; ** p < 0.01) and GAPDH was used as the loading control. ( G ) Western blot for phospho-paxillin for tumor protein extracts of each group normalized with beta -actin. (The whole western blot is shown in the Figure S4 ). ( H ) Analysis of bands by densitometry show significant differences in the groups * p < 0.05. ( I ) Western blot of PANC-1 tumor protein extracts reacted with an antibody for beta-catenin normalized with beta -actin. (The whole western blot is shown in the Figure S4 ). ( J ) Analysis of the bands by densitometry show a decrease in the intensity of the bands in the proglumide and combination groups although this did not quite reach statistical significance.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Insulin promotes focal adhesion formation in dissociated cells. (A): Plots showing 24-hour cell survival of insulin with integrin beta1 and alpha6 blocking antibodies compared with Insulin+TNNT2 antibody (mock) on matrigel (***, p < .001, n = 3). (B): Western blot showing the level of integrin beta1 and phosphorylated integrin beta1-788-789 2 hours after plating on matrigel-coated surface in base medium with or without insulin. Quantification is shown in Supporting Information Figure S4A . (C): Western blot showing the protein expression of IGF1R and phospho-IGF1R 2 hours after plating on matrigel in base medium with or without insulin. Quantification is shown in Supporting Information Figure S4B . (D): Western blot showing the protein expression of focal adhesion kinase (FAK), phospho-FAK-Y397, Paxillin, and phospho-Paxillin-Y118 2 hours after plating on matrigel in base medium with or without insulin. Quantification is shown in Supporting Information Figure S4C . (E): Immunofluorescent image showing Paxillin, FAK, and phospho-FAK-Y397 expression in focal adhesions 2 hours after plating on matrigel-coated surface with or without insulin. Scale bar = 20 mum. Quantification is shown in Supporting Information Figure S4E . (F): Immunostaining showing FAK (red) and phospho-INSR/IGF1R-Y1158-Y1162-Y1163 (green) expression in focal adhesions 2 hours after plating on matrigel-coated surface with or without insulin. Nuclei were stained with Hoechst (blue). Scale bar = 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Phosphorylated paxillin (Ser-272) is localized in spindle poles during mitosis. ( A ) Mitotic cells were plated on coverslips and were stained with antibodies against phospho-paxillin (pS272-paxillin, pY31-paxillin or pY118-paxillin) and alpha-tublin. Nuclei were stained using DAPI. pS272-paxillin, but not pY31-paxillin and pY118-paxillin, is localized in the spindle poles. Scale bar: 10 mum. Arrows point to spindle poles. ( B ) Z stack images of cells showing connection of microtubule spindle with pS272-paxillin in the poles. Scale bar, 5 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Cell-Matrix Adhesions Disassemble near the Contact upon Collision (A) Outline of colliding cells showed in zoom of contact area between two cells. Cells outlined in cyan and magenta. The cell labeled in cyan is expressing membrane RFP (mRFP) (red) and GFP-FAK (green). (B and C) Total area (B) and average size (C) of CMAs near the contact upon a collision. 0 min = first frame in contact. n = 15 cells. (B insert) Total area of CMAs near the contact as cells separate. 0 min = last frame in contact n = 3 cells. (D) Disassembly rate of CMAs as indicated. Leading edge, n = 24; contact, n = 27. (E and K) Immunocytochemistry on cells in contact against p-paxillin and vinculin (E) or cells expressing FAK-GFP (K) as indicated. Bottom: color indicating fluorescence intensity. (F) Schematic illustrating how regions of interest were defined for analysis. Contact region illustrated in blue and free edge in pink. (G-J, L, and M) Total area and length of CMAs using the markers indicated. n = 80 cells for (G) and (H); n = 76 cells for (I) and (J); n = 16 cells for (L) and (M). (N-Q) CMA polarity determined by ratio of total area (or length) of p-paxillin (N and O) or vinculin (P and Q) labeled CMAs in free edge over contact or leading edge over trailing edge. Scale bars, 20 mum. Line graphs and box graphs show mean, error: +- SEM. *** p