P shp2

Primary antibodies used for Western blotting were as follows: p-Tyr (sc-508), GAPDH (sc-32233), SHP2 (sc-7384), MCL-1 (sc-819) from Santa Cruz Biotechnology; p-EGFR (1068) (3777), p-ErbB2 (1248) (2247), ErbB3 (4754), p-ErbB3 (1289) (4791), p-ErbB3 (1328) (14525), p-ErbB4 (1284) (4757), p-Akt (308) (4056), p-Akt (473) (4060), p-Erk (202/204) (4370), p-P70S6K (389) (9205), p-4EBP1 (37/46) (2855), BIM (2933), BCL-xL (2764), p-P90RSK (380) (9341), p-S6 (235/6) (4858), p-S6 (240/4) (5364), c-Myc (5605), p-SHP2 (542) (3751), GAB1 (3232), p-GAB1 (627) (3233), p-GAB1 (659) (12745), β-ACTIN (4970), from Cell Signaling Technology. Secondary antibodies used were mouse IgG (GE Healthcare Life Sciences; NXA931) and rabbit IgG (GE Healthcare Life Sciences; NA934). Alpelisib and trametinib were purchased from AbMole Biosciences. RMC-4550 was purchased from Selleckchem. The SHP2 inhibitor (SHP099) for cell culture and in vivo experiments was purchased from MedchemExpress and AbMole Biosciences and was dissolved in DMSO at a stock concentration of 10 mmol/L for in vitro experiments. Human recombinant growth factors epiregulin (EREG) was purchased from Sigma-Aldrich (SRP3033) and neuregulin-1 (5898-NR) was purchased from R&D Systems.

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.

Western blotting was performed as described previously [50 (link)]. The antibodies used in this study included the following: mouse monoclonal antibodies against β-actin (1:1000), gp130 (1:500) from Santa Cruz Biotechnology, E-cadherin (1:3000) from BD Biosciences (San Jose, CA, USA), rabbit monoclonal antibodies against SHP2 (1:1000) from Santa Cruz Biotechnology, N-cadherin (1:1000), Vimentin (1:1000), Erk1/2 (1:1000), p-Erk (1:1000), and p-SHP2 (1:500) from Cell Signaling Technology. All primary antibodies were diluted with 5% BSA in tris buffered saline tween-20 (TBST), and the membranes were incubated at 4 °C overnight. The membranes were washed with TBST three times and then incubated with HRP-linked secondary antibodies (Santa Cruz, CA, USA) for 1 h, at room temperature. All Western blots were detected by electrochemiluminescence (Amersham Pharmacia Biotech, Aylesbury, UK). β-actin was used as the internal control.

Proteins were extracted from mouse ovaries or cells by protein lysate containing 10 µg/ml protease inhibitor and 10 µg/ml phosphatase inhibitor. Then, western blotting assays were performed as previously described. Immunoblotting was performed with primary antibodies against AKT (Santa Cruz Biotechnology, sc-8312), p-AKT (Cell Signaling Technology, #4060), Caspase-3 (Santa Cruz Biotechnology, sc-56053), cleaved Caspase-3 (Cell Signaling Technology, #9661), Bax (Cell Signaling Technology, #2772), Bcl-2 (Cell Signaling Technology, #2876), p-SHP2 (Cell Signaling Technology, #5431), SHP2 (Santa Cruz Biotechnology, sc-280), p85 (Cell Signaling Technology, #4292), and Cyclin D2 (Abcam, ab3087). β-Tubulin (Proteintech, 66240-1-Ig) was used as a control. The grayscale of the western blotting bands was quantified by ImageJ (NIH).
For co-immunoprecipitation analysis, proteins were extracted as described above. After centrifugation at 12000× g for 15 min at 4°C, the supernatants were co-incubated with SHP2 antibody at 4°C overnight followed by incubation with protein A/G agarose beads for another 4 h according to the manufacturer's protocols.

For WB analysis, A549 and HCC827 cells were incubated with ZWB90-3 (3 µM and 0.3 µM, respectively) or Emi-IgG4 (1.5 µM and 0.15 µM, respectively) for 72 h. Whole-cell lysates were prepared in radio-immunoprecipitation assay (RIPA) buffer (NCM, Suzhou, China) with a 1% protease and phosphatase inhibitors cocktail (NCM, Suzhou, China). Protein concentrations of cell lysates were quantified using a bicinchoninic acid protein (BCA) assay kit (Tiangen, China). The antibodies for c-MET, phospho(p)-c-MET, Gab1, p-Gab1, AKT, p-AKT, SHP2, p-SHP2, ERK1/2, p-ERK1/2, RAS, GAPDH, and β-actin were obtained from Cell Signaling Technology (CST, MA, USA), and the anti-α-tubulin antibody was purchased from Santa Cruz Biotechnology (CA, USA). Enhanced chemiluminescence (ECL) detection reagents (NCM, Suzhou, China) were used to develop the protein signals (Clinx Science Instruments, Shanghai, China).