Pmet tyr1234 1235

Total protein was extracted from the cell lines following the standard protocol, and 20–40 μg of protein was used for immunoblotting^{27 (link)}. The blots were cut prior to hybridization with antibodies during blotting. The primary antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) were as follows: pHER2-Tyr1221/1222 (#2249), pEGFR-Tyr1173 (#4407), EGFR (#2232), pHER3-Tyr1289 (#4791), pMET-Tyr1234/1235 (#3077), pIGF1R-Tyr1135/1136 (#3024), IGF1R (#9750), ERK (#4695), pAKT-Ser473 (#4060), pAKT-Thr308 (#4056), AKT (#9272), pH2A.X-Ser139 (#9718), cleaved PARP (#9541), and PCNA (#2586). Other primary antibodies [HER3 (sc-285), MET (sc-514148), pERK-Tyr204 (sc-7383), and PTEN (sc-6818)] were purchased from Santa Cruz Biotechnology, Inc. Anti-HER2 (ab16901) and anti-α-tubulin (T6199) antibodies were purchased from Abcam Inc. and Sigma-Aldrich, Inc., respectively. After incubation with peroxidase-conjugated secondary antibodies for 1 h at 20 °C, the protein blots were developed using an enhanced chemiluminescent reagent (Amersham Inc., Amersham, UK). Total protein visualized on the ChemiDoc™ XRS + System (Bio-Rad) and analysed on Image Lab software (Image Lab 6.0, Bio-Rad).

Total protein was extracted using a lysis buffer (Pierce, Rockford, IL, USA) and quantified with the Bradford method [40 (link)]. Fifty μg of the total protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). Immunoprecipitation assays were performed as described previously [41 (link)]. Membranes were incubated overnight at 4°C with the following primary antibodies: Lasp1 (1:100, Abcam, Cambridge, UK); GAPDH (1:5000, Sigma, St. Louis, MO, USA); Myc-tag, CyclinD1, CyclinB1, CyclinA2, CyclinE1, Snail, p-ERK, ERK, p-AKT-Ser473, AKT, p-p38, p38, p-FAK-Tyr397, p-FAK-Tyr925, p-FAK-Tyr576/577, FAK, p-Met-Tyr1234/1235, p-Met-Tyr1349, p-Met-Tyr1003, Met, p-Gab1-Tyr307, Gab1 (1:1000; Cell Signaling Technology, Danvers, MA, USA); E-cadherin(1:1000; BD Transduction Laboratories, Lexington, KY, USA); Zo-1 and Occludin(1:500; Proteintech, Chicago, IL, USA). PF-562271 was purchased from Selleck Chemicals (Houston, TX, USA), LY294002 was purchased from Cell Signaling Technology. Membranes were washed and subsequently incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology) at 37 °C for 2 hours. Bound proteins were visualized using electrochemiluminescence (Pierce, Rockford, IL, USA) and detected with a bio-imaging system (DNR Bio-Imaging Systems, Jerusalem, Israel).

Total cellular protein was extracted using RIPA lysis buffer supplemented with phosphatase and protease inhibitor cocktail and EDTA (Sigma-Aldrich, St. Louis, MO, USA). The protein samples (20–40 µg each) were subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Immunoblotting was performed using antibodies against MET (#4250S, Cell Signaling Technology, Danvers, MA, USA; diluted at 1:1000 in 5% skim milk), phospho-MET (p-MET) (Tyr1234/1235) (#3126S, Cell Signaling Technology, Danvers, MA, USA; 1:500 in 5% bovine serum albumin), PD-L1 (#1368S, clone E1L3N, Cell Signaling Technology, Danvers, MA, USA; 1:1000 in 5% skim milk), PD-L2 (#MAB1224-100, clone 176611, R&D systems, Minneapolis, MN, USA; 1:500 in 5% skim milk), or β-actin (#sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:5000 in 5% skim milk). The bound antibody was visualized using a chemiluminescence kit (Amersham Pharmacia Biotech, Uppsala, Sweden).

Paroxetine HCl, an anti‐depressant agent, was obtained from APExBIO (Houston, TX, USA). Antibodies for cleaved caspase‐8, cleaved caspase‐3, PARP, p‐EGFR (Tyr1068), p‐ERBB3 (Tyr1289 and Tyr1328), ERBB3, p‐ERBB2 (Tyr1248), ERBB2, p‐MET (Tyr1234/1235), MET, p‐AXL (Tyr702), AXL, p‐IGF‐1Rb (Tyr1131), IGF‐1Rb, p‐AKT (Ser437), AKT, p‐ERK (Thr202/Tyr204), ERK, p‐p38 (Thr108/Tyr182), p38, p‐JNK (Thr183/Tyr185), and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for Bcl‐2, GAPDH, and EGFR and the secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Halt protease and phosphatase inhibitor cocktail (100×), EDTA (100×), and bicinchoninic acid (BCA) protein assay kit were obtained from Thermo Fisher Scientific (Rockford, IL, USA). MTT and DMSO were obtained from Sigma‐Aldrich (St. Louis, MO, USA). PVDF membranes were purchased from Bio‐Rad (Hercules, CA, USA). SuperSignal West Dura Extended Duration Substrate was purchased from Thermo Scientific (Waltham, MA, USA). A human phospho‐RTK array kit was purchased from R&D Systems (Minneapolis, MN, USA).

Tissue were homogenized with a Polytron and cells were washed in PBS and lysed in M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific) containing the Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Protein concentration was quantified using the Pierce™ Microplate BCA Protein Assay Kit (ThermoFisher Scientific). Membranes were blocked in in 5% non-fat milk for 1 h and incubated with the following primary antibodies: β-catenin (1:2000; BD Transduction Laboratories™), p-β-catenin (Ser45; 1:1000; Cell Signaling Technology), Myc-tag (1:2000; MMCRI), Glutamine Synthetase (1:5000; BD Transduction Laboratories), c-Met (1:400, Invitrogen), p-Met (Tyr1234/1235; 1:1000; Cell Signaling Technology), cyclin D1 (1:10000; Abcam, Cambridge, UK), β-actin (1:5000; Sigma-Aldrich) and GAPDH (1:10000; EMD Millipore). Each primary antibody was followed by incubation with secondary antibody (1:5000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 h. After appropriate washing, bands were revealed with the Super Signal West Dura Kit (ThermoFisher Scientific).