Anti sos1

Immunoblotting was conducted as previously reported^{30 (link)}. Briefly, cells were washed twice in ice-cold PBS and lysed in lysis buffer [50 mM Tris (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 1× protease inhibitor (Roche) and PhosSTOP (Roche)]. Whole cell extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies: anti-RUNX1 (sc-365644, Santa Cruz Biotechnology, Inc.), anti-phosphorylated HER2 (#2243, Cell Signaling Technology), anti-HER2 (#2165, Cell Signaling Technology), anti-SOS1 (sc-10803, Santa Cruz Biotechnology, Inc.), anti-GAPDH (sc-47724, Santa Cruz Biotechnology, Inc.). For secondary antibodies, HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology) and anti-mouse IgG (#7076, Cell Signaling Technology) were used. Blots were visualized using Chemi-Lumi One Super (nacalai tesque, Inc.) and ChemiDoc^TM XRS + Imager (Bio-Rad Laboratories, Inc.) according to the manufacturer’s recommendations. We used total protein from human adult normal tissue (#P1234248, Biochain Inst., Inc.) as a normal control of stomach in Fig. 2c.

Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [50 mM Tris (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 1× protease inhibitor (Roche) and PhosSTOP (Roche)]. Whole cell extracts were processed for immunoprecipitation with the following antibodies at 4 °C overnight. anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology). Protein G Sepharose^TM 4 Fast Flow (17-0618-01, GE Healthcare) was added and an hour after incubation at 4 °C, the beads were washed three times in ice-cold wash buffer [20 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween 20]. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies: anti-HER2 (#2165, Cell Signaling Technology) and anti-SOS1 (sc-10803, Santa Cruz Biotechnology, Inc.). For secondary antibodies, HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology) was used. Blots were visualized using Chemi-Lumi One Super (nacalai tesque, Inc.) and ChemiDoc^TM XRS + Imager (Bio-Rad Laboratories, Inc.) according to the manufacturer’s recommendations.