44-626G

antibody from Invitrogen Antibodies

Targeting: PTK2 FADK, FAK, FAK1, PPP1R71

Provider product page for 44-626G

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Antibody data

Antibody Data
Antigen structure
References [48]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Other assay [34]

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Product number: 44-626G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-FAK (Tyr861) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated FAK. The final product is generated by affinity chromatography using a FAK-derived peptide that is phosphorylated at tyrosine 861.

PTK2 protein structure - 44-626G shown in red.

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Supportive validation

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Experimental details: Fig. 1 Mechanosignalling regulates melanoma cell adhesion and nuclear YAP/TAZ localization. a Morphology of 501mel and WM266-4 cells cultured for 72 h on collagen with the indicated stiffness degrees. A scale for matrix elasticity of tissues is shown varying from soft brain to rigid bone (adapted from [ 18 ]). Scale bar represents 20 um. b Quantification of cell morphology of 501mel and WM266-4 cells cultured for 72 h on collagen with the indicated stiffness degrees ( n = 3 experiments; n = 100 cells). Graphs show mean +- SEM. Tukey's post-test following one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. c Western blot analysis of pFAK, pSRC and pMLC2 of 501mel and WM266-4 cells cultured on collagen with the indicated stiffness degrees for 72 h. Total SRC, FAK and ERK2 was used as a loading control. d Immunofluorescence analysis of YAP/TAZ localization in 501mel cells cultured on collagen with the indicated stiffness degrees for 72 h. Scale bar represents 50 um. e Quantification of YAP/TAZ localization in 501mel and WM266-4 cells cultured on collagen with the indicated stiffness degrees for 72 h ( n = 3 experiments; n = 100 cells). f Western blot for LATS and YAP phosphorylation in 501mel cells cultured on collagen with the indicated stiffness degrees for 72 h. g Quantification of YAP/TAZ localization in 501mel cells cultured on collagen with the indicated stiffness degrees treated with 1 muM AZD0530 (SRCi), 1 muM PF562271 (FAKi) or DMSO for 24 h ( n = 3 experiments; n =

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Experimental details: Figure 4 The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions. Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Delta375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 um.

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Experimental details: Figure 4 GFP-LIMK1 fusions increase phosphorylation status of FAK signaling components . (A) Western blot analysis of whole cell lysates from MDA-MB-231 stable transductants probed with antibodies against pFAK, total FAK, pPaxillin, total Paxillin, pSrc, total Src, pAKT, total AKT, pErk1/2 and total Erk 1/2. (B) Western blot analysis for tubulin or GAPDH for each extract used in panel A, as loading controls.

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Experimental details: Figure 4. Tsp4b is required for muscle-specific integrin signaling at MTJ. ( A - F ) Lateral views of 20-24 hpf embryos stained with anti-phosphorylated (Tyrosine 861) FAK (pFAK; red) and anti-MHC (green). ( A and B ) pFAK localizes to the ends of early myofibers at wild-type somite boundaries. ( C and D ) Reduced pFAK levels in Tsp4b-deficient embryos. ( E and F ) pFAK levels are restored in Tsp4b-deficient embryos injected with full length tsp4b mRNA. ( G ) Fluorescence intensity measurements (arbitrary units [A.U.]) for pFAK staining along somite boundaries confirm significant reductions in Tsp4b-deficient embryos, and partial rescue by co-injection of full length tsp4b mRNA ( t test: one tailed, unequal variance; p-value: wt and tsp4b-deficient

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Experimental details: Figure 6 Western blot analysis of key signaling molecules implicated in calcium-dependent pathways and TNNC1 transcriptional regulation. Western blot analyses on proteins isolated from MFAP5-treated A224 and ALST ovarian cancer cells showed that, ( A ) Treatment of exogenous MFAP5 increased p-FAK (Y861), p-PLC-gamma1 (Y783), p-PKCtheta (T538), p-ERK1/2 (T202/Y204), p-CREB (S133), total Jun, and p-Jun (S63, S73 and S243) expressions in A224 ovarian cancer cells. ( B ) Treatment of exogenous MFAP5 increased p-FAK (Y861), p-PLC-gamma1 (Y783), p-PKCtheta (T538), p-ERK1/2 (T202/Y204), p-CREB (S133), total Jun, and p-Jun (S63, S73 and S243) expressions in ALST ovarian cancer cells. Relative normalized protein expression levels with respect to the corresponding control were presented. ( C ) A graphical summary of the molecular signaling events involved in MFAP5-mediated ovarian cancer cell motility and invasiveness. ( D ) The presences of FAK, ERK or CBP/CREB interaction inhibitors abrogated the motility promoting effects of MFAP5 in both A224 and ALST ovarian cancer cells (mean +- SD of 3 independent experiments; p < 0.001; two-tailed Student t -test).

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Experimental details: Figure 5 Kinetic analysis of FAK, PI3K p85 subunit of PI3 kinase and PDK1 phosphorylation in SK-MEL-28 melanoma cells after incubation with NC1(XIX) Western blot analysis of: ( A ) phosphorylated-FAK Y397 , ( B ) phosphorylated-FAK Y861 , ( C ) phosphorylated-PI3K p85 Y458 and ( D ) phosphorylated-PDK1 S241 compared to total FAK, total PI3K p85 and total PDK1 expression respectively after incubation of SK-MEL-28 melanoma cells with NC1(XIX) for 0, 1, 5, 15, 30 and 60 min. Bands were quantified by densitometric analysis. Phosphorylated protein was reported to corresponding total protein.

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Experimental details: Figure 3 Integrin beta1 is required for the invasion & metastasis of the murine mesenchymal cell lines. ( a ) Western blot analysis of FAK pathway activation in the mesenchymal vs epithelial cells lines of the murine cell line panel, and in cell lines stably expressing Zeb1 ( b ). ( c ) Western blot of FAK activation in the human H157 cells with inducible miR-200ab expression. ( d ) 393P_ZEB1 cells grown in Matrigel/Collagen I (1.75 mg/ml) and treated with an ITGbeta1-blocking antibody or IgM control for 7 days. ( e ) Quantitative RT-PCR and Western blot of 393P_ZEB1 cells after ITGbeta1-shRNA knockdown. ( f ) In vitro migration and invasion assay for the Itgbeta1 shRNA cells. *p < 0.02, **p < 0.002, ***p

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Experimental details: Figure 5 CRKL regulates FAK/Src complex formation at focal adhesions and the invasive/metastatic phenotype. ( a ) qRT-PCR and Western blot ( b ) analysis of 344SQ_shCRKL knockdown cells showed a decrease in FAK signaling. ( c ) CRKL knockdown decreased adhesion to fibronectin and 2D Transwell migration/invasion ( d ). ( e ) Immunofluorescent staining and biochemical fractionation ( f ) of CRKL knockdown cells for activated Src Y 418 , CRKL Y 207 , PaxY 118 , and p-FAK Y 861 in the focal adhesion complex at the membrane. An average of 20-30 cells was counted for the presence of focal adhesions and data are presented per cell. Scale bar is 200 mum. *p < 0.005, **p

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Experimental details: Fig. 7 Gain and loss of Ang2 in neural crest cultures affects the amount of phosphorylated FAK. Neural tubes electroporated with either a GFP control vector ( a ), Ang2-FL ( b ), or Ang2-shRNA ( c ) were plated on fibronectin/poly- l -lysine coated glass and allowed to grow for 24 h. Electroporated vector in blue . The cultures were then stained with HNK1 ( red ) and pFAK ( green ) antibodies. Scale bars 50 mum. Inset images in a , b , and c : individual filopodia in the pFAK channel showing the size measurement. Scale bar 3 mum. d The average pFAK cluster size. Each cluster of pFAK was individually measured by the spot detection algorithm (Imaris) based on the fluorescence in the pFAK channel, so thousands of clusters were used to calculate the average, SEM, and comparison between conditions. Control 4 neural tubes and n = 4179 pFAK clusters, Ang2-FL 4 neural tubes and n = 3182 pFAK clusters, 4 neural tubes and Ang2-shRNA n = 3298 pFAK clusters. All conditions are statistically different from each other with control vs. Ang2-FL p = 1 x 10 -22 , control vs. Ang2-shRNA p = 5 x 10 -85 , and Ang2-FL vs. Ang2-shRNA p = 1 x 10 -132

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Experimental details: Figure 1 FAK expression and phosphorylation during development. (A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1 st column), early gastrula (2 nd column) and late gastrula embryos (3 rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 um.

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Experimental details: Figure 4 The correlation of c-Src activity with an EMT. (a) Nuclear pStat-3 (blue) in invasive lobular carcinoma (ILC) cells with activated c-Src (red). Note the weak nuclear Stat-3 staining in lobular carcinoma in situ (LCIS; asterisk). An antibody against total Stat-3 was used as a control. (b) Left panel: widespread Stat-3 activation in ILC cells. Middle panel: increased levels of activated Fak in ILC cells surrounding nonneoplastic epithelium and LCIS (asterisks). Right panel: expression of pFak in LCIS (asterisk) and ILC (right). An antibody against total Fak was used as a control. (c) Colocalization of active c-Src (green) and active Fak (red) in ILC cells. (d) ILC cells with activated c-Src (green) are positive for the mesenchymal marker vimentin (red). (e) Expression of the mesenchymal N-cadherin (green) in a subset of invasive cancer cells.

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Experimental details: Figure 5 LIMK1 expression correlates with phosphorylation of FAK in focal adhesions of MDA-MB-231 cells . MDA-MB-231 cells expressing GFP-only or various GFP-LIMK1 fusions were fixed and stained with antibodies against phospho-FAK (red), and phalloidin against actin (gold). GFP fluorescence is green in this figure. Images were obtained via fluorescence microscopy (non-confocal). Cellular regions with focal adhesion structures are marked by white arrows in the phospho-FAK images. Scale bar represents 20 microns. Note: non-specific binding of secondary antibody stains nuclei red.

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Experimental details: Figure 3 FRNK does not act as a dominant negative in early Xenopus embryos. (A-D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E-H) Same as A-D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I-L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M-P) Same as I-L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphoryla

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Experimental details: Figure 3 Effects of vehicle and neuregulin treatment on selected kinases . A-D) Quantitation of phosphorylated proteins compared with respective total protein or GAPDH for each treatment group is shown. Sample number is indicated within each bar. Significance was determined by 1-way ANOVA and Newman-Keuls multiple comparison test using GraphPad software. ** P

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Experimental details: Figure 1 Dasatinib resistant cells exhibit differential sensitivity to the Src inhibitors, saracatinib and bosutinib (A) Dasatinib IC50 values of control and dasatinib-resistant (DasRes) cell lines that were calculated based on a growth curve with concentrations ranging from 0 - 10muM. (B-C) Sulfurhodamine B (SRB) growth assays were performed on the DasRes cell lines to determine sensitivity to saracatinib (B) and bosutinib (C). Three independent biological replicates were performed, and the standard error mean is displayed in the quantification graphs. (D-E) BRAF-mutant (D) and RAS-mutant (E) control and DasRes whole cell lysate was harvested after 24 hour treatments of DMSO, dasatinib, bosutinib, or saracatinib. Control cell lines were treated with 100nM dasatinib, and DasRes cell lines were treated with 2muM dasatinib. FAK/Src signaling was used to determine whether each inhibitor had similar efficacy at inhibiting Src activity. Three independent biological replicates were performed, and representative blots for signaling proteins and loading controls are shown. The pY416 Src blot was stripped and reprobed for total Src.

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Experimental details: Figure 3 The mTOR inhibitor, everolimus, inhibits cell growth and clonogenicity, but does not induce apoptosis (A) Cells were treated with DMSO or Everolimus for 72 hours, and cell number was counted using the ViCell Cell Counter. Fold changes are compared to DMSO. Three independent biological replicates were performed, and the standard error mean is displayed in the quantification graphs. p-value * = 0.05 - 0.01, phi = 0.01 - 0.001, delta = 0.001 - 0.001, Psi < 0.0001. (B-C) BRAF-mutant (B) and RAS-mutant (C) control and DasRes cells were treated with the indicated inhibitors for 24 hours. Cell lysate was harvested and a Western blot was performed to determine changes in the AKT/mTOR pathway signaling. Control cells were treated with 100nM dasatinib, and DasRes cells were treated with 2muM dasatinib. Three independent biological replicates were performed, and representative blots for signaling proteins and loading controls are shown. The pY416 Src blot was stripped and reprobed for total Src. (D-E) BRAF-mutant (D) and Ras-mutant (E) control and DasRes cells were treated with the indicated inhibitors for 7 days, and then released for 7 days to assess colony growth after inhibitor treatment. After 2 weeks, cells were fixed and stained with crystal violet. (F) Apoptosis was measured after 24 hours of everolimus treatment by caspase 3/7 cleavage using the Caspase-Glo 3/7 kit. Fold changes were calculated by comparing treatments to DMSO. Three independent biological

Supportive validation

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Experimental details: Figure 5 The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner. HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A-D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E-H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I-L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M-P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected contro

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Experimental details: Figure 1 Zeb1 expression induces EMT/miR-200 repression and FAK pathway activation in previously non-invasive cells. ( a ) Zeb1 expression causes a morphology change (middle panel) and is reversed upon re-expression of miR-200 (lower panel). Cells were stained with Integrin alpha6 (red), ZO-1 (green) and DAPI. ( b ) 393P cells with constitutive Zeb1 expression and 344SQ cells with constitutive miR-200 family expression grown on agarose and stained with phalloidin (red), DAPI and cortactin (green, lower panels). ( c ) Quantitative RT-PCR (left) and Western blot (right) analysis of EMT markers in the indicated cell lines. ( d ) Taqman RT-PCR for the miR-200 family members in the indicated cell lines. ( e ) In vitro Transwell migration and invasion assay for the 393P cell line panel. *p < 0.004, **p < 0.001 ( f ) Induction of miR-200a and b in H157 causes a morphology change. ( g ) Western Blot analysis of EMT markers in H157 + /- miR-200a and/or b. ( h ) In vitro Transwell migration and invasion assay for the inducible H157 cells. ( i ) Immunofluorescence of 393P_vec and 393P_ZEB1 cells stained for p-Src Y 418 , p-FAK Y 861 , and phalloidin (left); an average of 15-30 cells was counted for focal adhesions/cell with individual puncta considered a focal adhesion. Scale bar is 200 mum. (J) Western blot analysis of total cell lysates, cytosolic or membrane particulate fractions from 393P_vec and 393P_ZEB1 cells.

Supportive validation

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Experimental details: Figure 4 CRKL is a miR-200 target that regulates integrin-dependent signaling and is prognostic of patient outcome. ( a ) Schematic of the bioinformatic analyses applied to TCGA pan-cancer datasets to select potential miR-200b targets of relevance to lung cancer metastasis. From the top 5000 genes most anti-correlated with expression of miR-200b, we additionally considered genes positively correlated with ZEB1 expression (p < 0.0001, Pearson's), predicted miR-200b target genes (by TargetScan, miRanda, and miRDB), and genes associated with worse prognosis (p < 0.05, univariate Cox) in lung adenocarcoinomas 25 . Genes listed met all of the above criteria. ( b ) Heatmap showing the correlation of CRKL expression with the listed feature for each of the tumor specimens included from TCGA pan-cancer datasets by Pearson's coefficient. Significance of correlation: p < 1E-12 for miR-200c, p < 1E-35 all other features. ( c ) Kaplan-Meier plot of overall survival in patients from TCGA datasets, as stratified by CRKL expression. P-value by log-rank test. ( d ) qRT-PCR analysis of adaptor molecules in the paired epithelial and mesenchymal murine cell lines. ( e ) Western blot of total and phospho-CRKL expression in the murine cell lines and the genetically manipulated 393P cell lines. ( f ) Luciferase reporter assay in H157 cells using hRL_3' CRKL wt and mutant constructs. *p < 0.05, **p < 0.03, ***p < 0.003. The CRKL 3'UTR contains two predicted miR-200b/c/429 sites a

Supportive validation

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Experimental details: Figure 5 Effect of combination treatment with dasatinib and EC-8042 in FaDu xenografts. Mice with established FaDu xenografts were randomly assigned to four different treatment groups ( n = 10 tumors per group) and treated with saline buffer intravenously (i.v.) and/or tartaric acid (orally) (control), dasatinib (orally) at a dose of 10 mg/Kg every day (16 doses), EC-8042 (i.v.) at a dose of 50 mg/Kg every 7 days (4 doses), or the dasatinib plus EC-8042 combination (combo). Animals were sacrificed 4 h after the last treatment with dasatinib and 24 h after treatment with EC-8042. ( A ) Curves representing the mean tumor volume of FaDu xenografts during the treatments. Drug efficacy expressed as the percentage of tumor growth inhibition (%TGI) at the end of the experiment is indicated. Mean +- SD ( n = 10 per treatment group), RM-one-way ANOVA, Tukey's test, * p < 0.05, ** p < 0.01 vs. control (black) and dasatinib-treated mice (red). ( B , C ) Distribution of tumor volumes ( B ) and tumor weights ( C ) at the end of the experiment. Mean +- SD ( n = 10 per group), one-way ANOVA, Tukey's test, p < 0.05 between the indicated groups. ( D ) For the evaluation of CSC subpopulations after drug treatments, xenograft tumors were harvested and dissociated into single cells to assess the tumorsphere-forming ability after in vivo drug treatment. Mean +- SD ( n = 3 per group), one-way ANOVA, Tukey's test. ( E ) Representative images and ( F - J ) graphs of the immunohistochemical analysis