Cells were collected and lysed in RIPA (Beyotime, Jiangsu, China) supplemented with protease inhibitor cocktail (Roche, IN, USA) and then were incubated on ice for 30 min, followed by centrifugation at 12,000 g at 4°C for 30 min. Next, the BCA protein assay kit (Beyotime, Jiangsu, China) was used to determine the concentration of proteins. Equal total protein was loaded into each well for separation. After electrophoresis, the proteins were transferred to a nitrocellulose membrane and were blocked with 5% non-fat milk in TBST (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4, supplemented with 0.1% Tween 20) for 1 h. Next, primary antibodies (Src (#2018), p-Src (Tyr416) (#6943), FAK (#3285), p-FAK (Tyr397) (#8556), Paxillin (#2542), p-Paxillin (Tyr118) (#2541), GAPDH (#5174)) (Cell Signaling Technology Danvers, MA, USA) and Beta-actin (Abmart) were added and incubated at 4°C overnight. After washing with TBST, horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (KangCheng Bio-tech, Shanghai, China) was added and incubated for 1 h at room temperature. The membranes were washed and developed with enhanced chemiluminescence (ECL) (Pierce) and were subsequently exposed to KODAK X-OMAT BT Film (Kodak, Rochester, NY).
P fak tyr397
Manufactured by Cell Signaling Technology
Sourced in United States
P-FAK (Tyr397) is a phospho-specific antibody that detects focal adhesion kinase (FAK) phosphorylated at tyrosine 397. FAK is a key regulator of cell adhesion and signaling.
Automatically generated - may contain errors
Lab products found in correlation
P akt ser473, by Cell Signaling Technology (5 mentions)
P src tyr416, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
P mtor ser2448, by Cell Signaling Technology (2 mentions)
P fak, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Paxillin, by Cell Signaling Technology (1 mentions)
P paxillin tyr118, by Cell Signaling Technology (1 mentions)
X omat bt film, by Kodak (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Proteinase and phosphatase inhibitor, by Roche (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Rock1, by Santa Cruz Biotechnology (1 mentions)
P ampk thr172, by Cell Signaling Technology (1 mentions)
Gapdh abs16, by Merck Group (1 mentions)
25 protocols using p fak tyr397
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Analysis in Ventricular Tissue
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Protein samples were prepared as previously described [14 (link), 33 (link), 36 (link)]. Ventricular tissue fragments were disrupted with a PYREX® Potter-Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche). The homogenate was centrifuged at 15,000 x g at 4°C for 15 minutes, and the supernatant was saved for immunoblotting. The blots were probed with primary antibodies to ROCK2 (sc-5561), FAK (sc-558) from Santa Cruz Biotechnology, ROCK1 (#4035), p-FAK-Tyr397 (#3283), Bax (#2772), LC3 (#2775), Beclin 1 (#3738), p62 (#5114) and p-AMPK-Thr172 (#2535) from Cell Signaling Technology, and p-Beclin1-Thr119 (ABC118) from MilliporeSigma. All blots were normalized to GAPDH (ABS16; MilliporeSigma) or to actin (MABT523; MilliporeSigma).
3
Investigating Effects of SPARC on Cell Adhesion
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Cancer cells were seeded onto cell culture dishes coated with collagen I. In addition, either the collagen substrate or the culture medium was supplemented with 750 ng/mL rSPARC or equal amounts of PBS + 0.1% BSA as control. After 24 h incubation, cells were lysed, and proteins extracted for western blotting as described in [43 (link)]. Briefly, protein was extracted using HEPES lysis buffer supplemented with protease inhibitors cocktail (Complete, Roche, Germany) and phosphatase inhibitors. 10 µg protein per lane were analyzed by western blotting. The antibodies used recognized integrin β1 (1:1000; 4706, Cell Signaling Technology, Danvers, MA, USA), FAK (1:1000; 71,433, Cell Signaling Technology), p-FAKTyr397 (1:1000; 8556, Cell Signaling Technology), E-cadherin (1:1000; 3195, Cell Signaling Technology), and β-actin (1:5000, AC-15, Sigma, St. Louis, MO). Detection was achieved by chemiluminescence and band intensity was determined using ImageJ software. Uncropped scans of all blots can be found in Figure S5 .
4
Integrin-mediated Signaling Pathway Analysis
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HGEs were digested using trypsin, and the obtained cells were seeded on material surfaces of four groups (ppAA, ppAC, ppME and Control) at a density of 106 cells/well in cell incubator for 1 h. The samples were gently rinsed twice with PBS. The total RNA from HGEs was extracted to detect the expression levels of PI3KCA, PI3KCB, mTOR, AKT1, AKT2, PTEN, Eif4A, Eif41B, Eif4G, RPS6K, PDK1, 4EBP (Table S2 ). Western blot was used to detect the expression of adhesion-related proteins, including P-FAK (Tyr397, 1:1000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology) and P-AKT (Thr308, 1:1000, Cell Signaling Technology), mTOR (1:1000, Cell Signaling Technology) and P-mTOR (Ser2448, 1:1000, Cell Signaling Technology). The cells were further cultured on ppAA chemical surface with or without FN12-8 (FN inhibitor, Takara) and mAb13(ITGβ1 inhibitor, Cell Signaling Technology), and underwent the above-mentioned western blot assay to detect the protein expression of Integrin β1, FAK, P-FAK, mTOR and P-mTOR.
5
Western Blot Protein Analysis
6
Signaling Pathway Analysis in Cancer Cells
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Cell culture medium was purchased from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Atlas Biologicals (Fort Collins, CO). Primary antibodies targeting the following were as detailed: Actin, EGFR, pEGFRTyr1069, pEGFRTyr1173, pEGFRTyr1110, ERK1/2, pERK1/2Thr202/204, mTOR, pmTORSer2441, pFAKTyr397, pFAKTyr527, pFAKTyr925, FAK, pMEKSer221, MEK, IKKα/β, pIKK α/βSer180/181, pSRCTyr418, SRC, pAKTSer473, AKT, pSTAT1Ser727, STAT1, Hsp70 and PLCγ were from Cell Signalling (Danvers, MA). Antibodies specific for BAG3 were from Abcam (Cambridge, UK). Secondary HRP-conjugated antibodies, including anti-rabbit Gig and anti-mouse IgG were from Cell Signalling (Danvers, MA). YM-1 was obtained from Sigma Aldrich and the compounds 5-carbamimidamido-2-[2-(phenylformamido) acetamido pentanoic acid (ZINC02516109) and 2-(quinoline-3-carbonyl)-2,8-diazaspiro[4.5]decane-3-carboxylic acid (ZINC72169376) sourced from MolPort (Europe).
7
Antibodies for Protein Signaling Analysis
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We used the following antibodies in this study: STIM1 antibody for immunohistochemistry (MA1-19451; Thermo Fisher Scientific), STIM1 antibody for Western blotting (mouse monoclonal; 610954; BD), Orai1 (rabbit polyclonal; O8264; Sigma-Aldrich), GAPDH (mouse monoclonal; G8795; Sigma-Aldrich), Src (mouse monoclonal; clone GD11 05-184; EMD Millipore), p-Src (Tyr416) (rabbit polyclonal; 2101; Cell Signaling Technology), Akt (mouse monoclonal; 2966; Cell Signaling Technology), p-AKT (Ser473) (rabbit monoclonal; 193H12; Cell Signaling Technology), FAK (rabbit polyclonal; 3285; Cell Signaling Technology), p-FAK (Tyr397) (rabbit polyclonal; 3283; Cell Signaling Technology), Tubulin (mouse monoclonal; T6199; Sigma-Aldrich), cortactin (mouse monoclonal; clone 4F11 05-180; EMD Millipore), and p230 trans-Golgi (mouse monoclonal; 611281; BD).
8
Antibody-Based Signaling Pathway Analysis
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Antibody against DGKα (Cat# H00001606–B01P) was from Abnova (Taipei, Taiwan, China). Antibody against FAK (N-terminal, Cat# sc-557) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against pFAK Tyr397 (Cat# 3283S), Janus kinase 2 (JAK2, Cat# 3230), phosphoJAK2 (pJAK2) Tyr1007/1008 (Cat# 3771), Janus kinase 3 (JAK3, Cat# 8827), pJAK3 Tyr980/981 (Cat# 5031), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cat# 5174), protein kinase B (AKT, Cat# 4691), pAKT Ser473 (Cat# 4060), and pFAK Tyr397 (Cat# 3283S) were from Cell Signaling Technology (Danvers, MA, USA). Z-Val-Ala-Asp-FMK (Z-VAD-FMK, Cat# S7023), and PF562271 (Cat# S2890) were from Selleck Chemicals (Houston, TX, USA). All flavonoids used in the present study were from MedChemExpress (MCE) Inc. (Shanghai, China).
9
Corosolic Acid Inhibits VEGFR2 Signaling
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Corosolic acid, ursolic acid, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and sulphorhodamine (SRB) were obtained from Sigma-Aldrich. Sorafenib was purchased from Santa Cruz Biotechnology. Lipofectamine 2000, VEGFR2 (KDR) siRNA, phalloidin, and Alexa Flour Dyes were obtained from Invitrogen Life Technologies. The primary antibodies against VEGFR2, p-VEGFR2 (Tyr1054), p-VEGFR2 (Tyr951), Src, p-Src (Tyr416), FAK and p-FAK (Tyr397) were purchased from Cell Signaling Technology. The Matrigel Matrix was obtained from BD Biosciences.
10
Signaling Pathway Investigation Protocol
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Antibodies against PLCγ1, p-PLCγ1 (Tyr783), Caspase-3, AMPKα, p-AMPKα (Thr172), ERK1/2, p-ERK1/2 (Thr202/Tyr204), mTOR, p-mTOR (Ser2448), p-ULK1 (Ser757), FAK, and p-FAK (Tyr397) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Antibodies against P62, Bcl-2, and ULK1 were purchased from Abcam Inc. (Cambridge, MA, USA). Antibody against LC3B was purchased from Novus Biologicals, Inc. (Littleton, CO, USA). Goat anti-Rabbit IgG and Goat anti-Mouse IgG were purchased from Protein Tech Group (Chicago, IL, USA). The dilution concentrations of anti-Rabbit and anti-Mouse were 1:50000 and others were 1:1000. PLCγ1 inhibitor U73122 (U), autophagy inhibitor PI3K inhibitor 3-Methyladenine (3-MA) and Chloroquine (CQ), MEK inhibitor PD98059, mTOR inhibitor Rapamycin, and AMPKα activator Metformin were purchased from Sigma-Aldrich in China (Shanghai, China). Caspase inhibitor Z-VAD-FMK (Z-VAD) and RIP1 inhibitor Necrostatin-1(Nec-1) were purchased from MedChemExpress USA (Monmouth Junction, NJ, USA). Antibodies against Ki67 and MMP2 were purchased from Ruiying Biological (Suzhou, China) and ABclonal (Wuhan,China) respectively. Other reagents were of the highest grade commercially available.
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