P met tyr1349

Proteinase, saline, dimethyl sulphoxide (DMSO), and MG132 were purchased from Sigma-Aldrich. Poly(I:C), mainly consisting of low molecular weight species (100b–500b > 90%), and LPS was purchased from Sigma-Aldrich. HGF recombinant human protein was purchased from Thermo Fisher Scientific (Waltham, MA). SHP099 was purchased from Cayman Chemical (Ann Arbor, MI). Primary antibodies to RIG-I (#3743), MDA5 (#5321), p-IRF3 (#83611), IRF3 (#4302), p-MET (Tyr1234/1235) (#3077), p-MET (Tyr1349) (#3121), MET (#3127), p-GAB1 (#12745), GAB1 (#3232), p-SHP2 (#5431), SHP2 (#3397), p-ERK (#9101), ERK1/2 (#9102), DC-SIGN (#13193), DYKDDDDK Tag (FLAG) (#14793), Myc (#2276), GAPDH (#2118) and β-actin (ACTB; #4970) were purchased from Cell Signaling Technology (Beverly, MA); phosphotyrosine (05-321) and K48-ubiquitin (05-1307) were from Merck Millipore (Burlington, MA); PRL-1 (PTP4A1) (ab168643) and LECT2 (ab119429) were from Abcam (Cambridge, UK); and p-MET (Tyr1356) (PA5-40218) was from Thermo Fisher Scientific. All antibodies were used at a 1:1000 dilution.

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).