P erk1 2 t202 y204 9101

Cells were washed twice in cold PBS and lysed with Laemmeli sample buffer. Lysates were resolved by SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. For secreted NRG1 detection, medium was collected and centrifuged for 30 mins at 4000 rpm in an Amicon ultra conical tube. Laemmeli sample buffer was added and samples resolved by SDS-PAGE. After blocking in 5% BSA, membranes were incubated with the indicated primary antibodies overnight at 4 °C, followed by incubation with peroxidase-coupled secondary antibodies. Bound antibodies were detected using enhanced chemiluminescence substrate (Pierce, Rockford IL). Chemiluminescence was visualized on a VersaDoc Multi-Imager and quantitated using Quantity-One software (BioRad, Hercules, CA). Primary antibodies used were: NRG1 (# MAB377) antibody purchased from R&D Biosystems (Minneapolis, MN) used for detection of secreted NRG1, ERK2 (sc-1647) and EGFR (sc-03) antibodies purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); NRG1 (#2573), p-ErbB3 (Y1197, #4561), ErbB3 (#4754), p-ErbB2 (Y1196, #6942), ErbB2 (#4290), p-AKT (S473, #6942), p-AKT (T308, #2965), AKT (#9272) and p-ERK1/2 (T202/Y204, #9101) purchased from Cell Signaling Technology; actin (A2066) antibody purchased from Sigma-Aldrich Co. (St. Louis, MO).