Western blotting analysis was performed as described in our previous study 24 (link). The following primary antibodies were used: rabbit polyclonal anti-FAK (pY397, 1:1000; Invitrogen) and rabbit polyclonal anti-β-actin (1:2000; Cell Signaling Technology, Beverly, MA, USA). Target protein signals were detected with a Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.
Anti py397 fak
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-pY397-FAK is a primary antibody that specifically recognizes the autophosphorylated tyrosine 397 residue of Focal Adhesion Kinase (FAK). This antibody can be used to detect the activated state of FAK in various cellular and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti fak, by Cell Signaling Technology (3 mentions)
Anti fak, by BD (2 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti adam10, by Abcam (1 mentions)
Irdye 800cw secondary antibody, by LI COR (1 mentions)
Anti egfr, by BioLegend (1 mentions)
Pe conjugated anti adam10, by BioLegend (1 mentions)
Tgx gradient gel, by Bio-Rad (1 mentions)
Anti paxillin, by Santa Cruz Biotechnology (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Tcs sp mp inverted confocal microscope, by Leica (1 mentions)
Triciribine, by Selleck Chemicals (1 mentions)
11 protocols using anti py397 fak
1
Quantitative Western Blot Analysis
2
FAK Activity Analysis by Western Blotting
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Western blotting analysis of FAK activity was conducted as described elsewhere (Puig et al., 2015 (link)). In brief, fibroblasts were lysed in extraction buffer supplemented with phosphatase and protease inhibitors. Equal protein amounts were separated on 10% Mini-PROTEAN TGX precast gels (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare), blocked, and incubated overnight with suitable primary antibodies including anti-FAK (3285; Cell Signalling), anti-FAKpY397 (700255; Invitrogen), anti–α-tubulin (5174 and 2144, Cell Signalling), or anti–β-actin (Sigma). The latter two markers were used as loading controls. Protein bands were labeled and visualized by chemiluminescence in a digital imaging system (ImageQuant LAS 4000; GE Healthcare). Protein band intensities were quantified with ImageJ and normalized to the corresponding loading control.
3
Quantitative Western Blot Analysis
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Primary antibodies used in this study were anti-ADAM10 (abcam #ab1997), PE conjugated anti-ADAM10 (Biolegend #352704), anti-v-Src (Calbiochem #OP07), anti-Src family pY416 (CST #2101), anti-MAPK14 (CST #9212, anti-MAPK14 pT180/pY182 (CST #9211), anti-FAK (BD #610088), anti-FAK pY397 (Biosource #44-625G), anti-FAK pY576 (Santa Cruz #sc-16563), anti-FAK pS910 (Biosource #44-596G), anti-MAPK3/1 (CST #9102), anti-MAPK3/1 pT202/pY204 (CST #9106), anti-PAK2 (CST #2608), anti-PAK1/2 pS144/pS141 (CST #2606), anti-BCAR1 (Santa Cruz #sc-860), anti-BCAR1 pY249 (CST #4014), anti-Paxillin (Santa Cruz #sc-5574), anti-Paxillin pY118 (CST #2541), anti-Shc pY239/pY240 (CST # 2434), anti-Gab1 pY627 (CST #3231), PE-conjugated anti-E-cadherin (BioLegend #324106), anti-EGFR (Biolegend #352904), anti-Vinculin (Life Technologies #700062).
Protein extracts were separated by one-dimensional SDS gel electrophoresis using 4–15% TGX gradient gels (Biorad) and subsequently transferred on a PVDF membrane (Merck-Millipore). Membranes were blocked for 1.5 h in 5% (w/v) dried milk in PBS-0.05% (v/v) Tween-20 and probed with various phospho-site specific primary antibodies and their corresponding pan antibodies overnight at 4°C. Detection was performed using either IRDye680RD or IRDye800CW secondary antibodies (LI-COR) and the Odyssey infrared imaging system (LI-COR).
Protein extracts were separated by one-dimensional SDS gel electrophoresis using 4–15% TGX gradient gels (Biorad) and subsequently transferred on a PVDF membrane (Merck-Millipore). Membranes were blocked for 1.5 h in 5% (w/v) dried milk in PBS-0.05% (v/v) Tween-20 and probed with various phospho-site specific primary antibodies and their corresponding pan antibodies overnight at 4°C. Detection was performed using either IRDye680RD or IRDye800CW secondary antibodies (LI-COR) and the Odyssey infrared imaging system (LI-COR).
4
Visualizing Phospho-FAK in HUVECs
5
Immunoprecipitation and Western Blot Analysis
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Anti-ARHGEF5 was generated in rabbits via immunization with GST-mouse or human ARHGEF5 (amino acids 2–204) and affinity purified using a maltose-binding protein-tagged antigen. Anti-Src-pY418, anti-GFP, anti-FAK-pY397, SD208, Alexa Fluor 488 phalloidin, Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G, horse radish peroxidase-conjugated goat anti-rabbit immunoglobulin G, anti-mouse immunoglobulin G, anti-occludin and anti-cortactin-pY421 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH, anti-Fyn, anti-Lyn and anti-vimentin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-v-Src, anti-cortactin (4F11) and anti-phosphotyrosine (4G10) were from Millipore (Billerica, MA, USA). Anti-FLAG (M2) and anti-β-tubulin were from Sigma-Aldrich (St Louis, MO, USA). Anti-E-cadherin, anti-N-cadherin and anti-FAK were from BD Transduction Laboratories (Lexington, KY, USA). anti-Smad2-pS465/467, anti-Smad2, anti-MLC2-pT18/S19, anti-MLC2, anti-Akt and anti-Akt-pS473 were from Cell Signaling Technology Inc. (Beverly, MA, USA). TGF-β1 and TNF-α were from PeproTech (Rocky Hill, NJ, USA). The Akt inhibitor triciribine was from Selleckchem (Houston, TX, USA).
6
Antibody and Reagent Use for Cell Culture
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The following antibodies and reagents were used for cell culture experiments: anti-vinculin (Sigma-Aldrich, V9131; IF; 1:200), anti-FAK (pY397) (Thermo Fisher Scientific, 44-624G; IF; 1:400), anti–integrin β1 (9EG7) (BD Pharmingen, 550531; IF; 1:200), anti–p-tyrosine (4G10) (Millipore, 05-1050X; IF; 1:200), anti-mouse immunoglobulin G (IgG) Alexa Fluor 405 (Thermo Fisher Scientific, A31553; IF; 1:200), anti-mouse IgG Alexa Fluor 647 (Thermo Fisher Scientific, A21235; IF; 1:500), anti-rabbit IgG Alexa Fluor 647 (Thermo Fisher Scientific, A21244; IF; 1:500), anti-rat IgG Alexa Fluor 647 (Thermo Fisher Scientific, A21247; IF; 1:500), CellMask Deep Red plasma membrane stain (Thermo Fisher Scientific, C10046; Live stain; 1:1000), collagen I (Corning, 354236), fibronectin (Merck, 341631), puromycin (Sigma-Aldrich, P8833), and hygromycin B (Sigma-Aldrich, H3274).
7
Immunofluorescence and Western Blotting Assay
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For immunofluorescence, anti-cortactin (ab-33333) was obtained from Abcam; anti–pY402-Pyk2 and anti–pY397-FAK were obtained from Invitrogen; anti-Arp2 (H-84; SC-15389) and anti-Tks5 (FISH M-300; SC-30122) were obtained from Santa Cruz Biotechnology, Inc.; and antivinculin (clone hVIN1; V9131), anti–pY421-cortactin (C0739), and anti–pY466-cortactin (C0864) were obtained from Sigma-Aldrich. Rhodamine-labeled phalloidin and Alexa Fluor–conjugated secondary antibodies were obtained from Molecular Probes. For Western blotting, anticortactin (clone 4F11; 05-180) and antiphosphotyrosine (clone 4G10; 05-321) were obtained from EMD Millipore; anti-Pyk2 (3480 and 3292) was obtained from Cell Signaling Technology; anti-FAK (clone 77; 610088) was obtained from BD; anti-GFP (clones 7.1 and 13.1; 11814460001) was obtained from Roche; and anti–β-actin (clone AC-15; A5441) was obtained from Sigma-Aldrich. Secondary antibodies (goat anti–mouse 680LT and goat anti–rabbit 800CW) were obtained from LI-COR Biosciences.
8
Intracellular Staining and Flow Cytometry
9
Flow Cytometric Analysis of ALDH Activity
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Flow cytometry was carried out as previously described [68 (link), 69 (link)]. For ALDH staining, cells from were stained with the Aldefluor reagent (Stem Cell Technologies) for 30 minutes in a 37°C water bath according to the manufacturer’s protocol. ALDH+ cells were identified by based on cells stained with Aldefluor in the presence of diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH, used to control for background fluorescence. For intracellular staining, cells were fixed with 2% formalin for 10 minutes, followed by permeabilization with 1% NP40 for 10 minutes at room temperature. Cells were stained with rabbit monoclonal anti-pY397-FAK (Invitrogen), rabbit polyclonal anti-FAK (Cell Signaling), or mouse monoclonal anti-ALDH (BD Biosciences) antibodies followed by anti-rabbit-APC and anti-mouse-FITC antibodies (Invitrogen). Annexin V staining was done according to manufacturer’s protocol (eBioscience).
10
Monoclonal Antibody Anti-CIB1 Protocol
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The monoclonal antibody anti-CIB1 was generated in our laboratory as described [16 (link)]. Purified human Fg was purchased from Enzyme Research Laboratories, Inc (South Bend, IN); BAPTA-AM, DMSO, U73122, an inhibitor of phospholipase C (PLC) and cytochalasin D were from Sigma (St. Louis, MO). Polyclonal antibodies against FAK, c-Src, and αIIb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- pY418Src and anti-pY397FAK was obtained from BioSource, (Carmillo, CA). Additionally, anti-αIIb (SEW8) was a generous gift from Dr. Peter J. Newman (Blood Research Center, Milwaukee, WI). PP2 was obtained from Calbiochem (San Diego, CA). Sequence and synthesis of αIIb cytoplasmic domain peptide and the corresponding scrambled peptide were described [18 (link)]. Other chemicals, unless indicated, were of analytical grade purchased from Sigma.
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