C myc 5605

Cells were washed with ice-cold PBS and then lysed with RIPA lysis buffer (150 mmol/L Tris, pH 7.4, 100 mmol/L NaF, 120 mmol/L NaCl, 100 µmol/L sodium orthovanadate, and 1× protease inhibitor cocktail and phosphatase inhibitor cocktail; Roche). Lysates (30 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes; which were incubated with primary antibodies at 4°C overnight or at room temperature for 2 hours followed by incubation with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) for 1 hour at room temperature. Immunoreactive bands were visualized by chemiluminescence (Santa Cruz Biotechnology). All primary antibodies were from Cell Signaling Technology with the exception of BRD4 (Abcam), Actin (Abcam) and AXL (Life Technologies). For phospho-RTK arrays (R&D Systems), lysates were procured and applied to arrays according to manufacturer’s instructions. Arrays were visualized using chemiluminescence. Antibodies used for immunoblotting were from Cell Signaling Technology (P-MET-Y1234 #3126, P-CDK9 #2549, P21 #2947, HER3 #12708, P-HER3-Y1289 #4791, ROR2 #88639, HER2 #2242, P-SFK-Y419 #6943, C-MYC #5605, P-MAPK #9101, GAPDH #5174), Santa Cruz Biotechnology (V5 probe #sc-271944), R&D Systems (P-AXL-Y779 #AF2228, AXL #AF154), Abcam (BRD4 #128874, c-MET #59884, Actin #6276, Beta-tubulin #6046) and BD Biosciences (EGFR #610016).