44-624G

antibody from Invitrogen Antibodies

Targeting: PTK2 FADK, FAK, FAK1, PPP1R71

Provider product page for 44-624G

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [177]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Other assay [20]

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Product number: 44-624G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-FAK (Tyr397) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Chicken/Avian, Drosophila, Xenopus
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Storage: -20°C

PTK2 protein structure - 44-624G shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Proline-rich tyrosine kinase 2 phosphorylation induced by acute stretch is inhibited by PF-4594755 (PF). Rat portal veins were cultured with either DMSO (0.08%) or PF (1 mu mol/L) overnight (A-D). Following overnight equilibration, portal vein strips were stretched by a hanging weight for 10 min and quickly frozen in liquid nitrogen. Phosphorylation of PYK2 (A), Akt (B), FAK (C), and ERK1/2 (D) was determined with phospho-specific antibodies and the signal intensity normalized to the signal intensity of the respective total protein ( n = 7-10). * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant, for comparison with unloaded strips.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 17-AAG and EC154 suppress UMRC2 cell motility and differentially modulate ERK, FAK, and src activation . ( a ) Serum starved UMRC2 cells were plated in transwell Boyden chambers for 16 h with a serum gradient of 0.1% (top) to 10% (bottom) in the presence of serially diluted concentrations of the indicated Hsp90 inhibitors. Migrating cells were fixed with 3.7% formaldehyde, stained with Crystal Violet and counted, with standard deviation shown. All drug treatments significantly inhibited migration in both cell types (*), as determined by ANOVA and Student's t- test ( p < 0.05). ( b ) UMRC2 and 786-O cells were treated for 20 h with the indicated doses of inhibitors and phosphorylated and total ERK, FAK, and src were analyzed by SDS-PAGE and Western blot.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 FAK is heavily phosphorylated in mesodermal tissues and integrin-free regions of cells. (A) Intensity color coded confocal section of the dorsal lip region from a whole mount immunostained gastrula stage embryo using a P-Y576 FAK antibody. Mesodermal cells (white arrow) display much higher levels of phospho-FAK than endodermal cells lining the forming archenteron (white arrowheads) and the endodermal cells of the blastopore (red arrow). (B) Same as A but showing the anterior mesendoderm and the animal cap from a whole mount immunostained gastrula stage embryo. The superficial cells of the animal cap (white arrowhead) show lower levels of phospho-FAK signal compared to deep cells (white arrow) and mesendodermal cells (red arrow). (C) High magnification color coded narrow optical section of superficial cells of the animal cap reveals that the apical surface of these cells display similar levels of phospho-FAK compared to the basolateral region while (D) the apical region of the deep cells of the animal cap facing the fibronectin ECM display significantly elevated levels of phospho-FAK compared to the basolateral region. In addition, in the deep cells of the ectoderm labeling of phospho-FAK in the basolateral region is relatively uniform but the apical region displays distinct foci of higher signal intensity (E-G) Confocal optical sections from whole mount immunostained embryos using integrin-beta1 (green) and P-Y397 FAK antibodies (red). Integrin-beta1 and P-Y397 FAK c

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

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Experimental details: Figure 6. ( A ) Survival outcome analysis in NSCLC patients with wild-type versus mutated PXN. Left and right graphs show overall and individual mutant survival curves, respectively. The relative expression of PXN, p-PXN, BCL-2, FAK, phospho-FAK, Cox IV, DRP-1, and MFN-2 was evaluated by IHC on two sets of samples; seven NSCLC patient samples bearing wild-type PXN and six NSCLC patient samples that were identified to have various PXN mutations as shown in ( B ). ( C and D ) represent the averaged staining intensity scores among all WT and all mutants. ( E ) Shows graph of staining intensity scores of the proteins assayed as expressed in the cytoplasm of the cells. ( F )The panel shows photographs of IHC stains represented in one wild-type and one mutant sample.

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

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. Reversibility of the effects of SB in A172 cells. (A) At 4 days after treatment with 2 mM SB, A172 cells were washed in media without SB and cultured for 4 additional days. The effects on the expression of p21 (FK506 binding protein like), p27 (cyclin dependent kinase inhibitor 1B), p53, SIRT1, pY397/pY577 FAK and p-MLC20 were determined by western blotting. (B) Cell motility was measured in A172 cells treated as in (A). Results are presented as the mean +- standard deviation (n=6). *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Western blotting was used to demonstrate that FAK-Src signaling is activated by si-p53. FAK, focal adhesion kinase; Src, Src proto-oncogene, non-receptor tyrosine kinase; si-p53, small interfering RNA targeting p53; p53, tumor protein p53; ERK, extracellular-regulated kinase; p-, phosphorylated; JNK, janus kinase; P-925, Phospho-FAK Tyr925; P-577, Phospho-FAK Tyr577; P-397, Phospho-FAK Tyr397.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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