Perk1 2t202 t204

Cultured cells were rinsed three times with ice-cold PBS, harvested, and lysed directly in RIPA buffer (Pierce, Thermo Scientific, Rockford, USA) for immunoblot analysis. Cell debris was removed by centrifugation at 15,000g for 10 minutes at 4°C. The supernatant was used as total protein lysate. For each sample, 10 μg of total protein lysate was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS page), followed by immunoblot analysis. Immunoblots were probed with antibodies against MYC (Y69, #ab32072; Abcam, Cambridge, UK), GAPDH (#G9295, Sigma Aldrich GmbH, München, Germany), Vinculin (hVIN-1, #9131, Sigma Aldrich GmbH), pERK1/2^T202/T204 (#9101, Cell Signaling Technology, Danvers, MA, USA), and ERK1/2 (C14, #SC-154, Santa Cruz Biotechnology, USA). All antibodies were used according to the manufacturer’s instructions. The results were visualized with secondary antibodies (GE Healthcare Life Sciences Europe, Freiburg, Germany) against mouse (NA9310) or rabbit (NA9340) primary antibodies combined with enhanced chemiluminescence (ECL) western blotting substrates (Pierce, Thermo Scientific).