Anti ereg

Cells were seeded in 60 mm plates and 48 h after transfection they were lysed with RIPA buffer (Sigma-Aldrich) containing Complete mini protease and phosphatase inhibitor cocktail tablets (Roche, Basel, Switzerland). Protein quantification was performed using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) and 10 µg of lysate was loaded per lane. Proteins were resolved by 8 or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and wet transferred to polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The signals were visualized by ECL Prime Western blotting Detection Reagent (Amersham, Piscataway, NJ, USA) and exposed to AGFA Curix X-ray film (AGFA, Mortsel, Belgium). The following Ab were used: anti-HOXB9 (1:100, mouse, sc-398500) from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and anti-EREG (1:1000, rabbit, 12048 S), anti-PARP (1:1000, rabbit, 9542 S) and anti-β-tubulin (1:2000, rabbit, 2146 S) from Cell Signaling Technology (Danvers, MA, USA).

Total protein extraction and SDS-polyacrylamide gel electrophoresis procedures were performed as described in a previous study [22 (link), 23 (link)]. Immune complexes on membranes were incubated with horseradish peroxidase‐conjugated anti‐ rabbit or anti‐mouse IgG (Promega, Madison, WI) and visualized with SuperSignal reagents (Pierce, Rockford, IL). The primary antibodies used in this study were anti‐EREG (Cat No. 93815; Cell Signaling Technology, Boston, MA, USA), mouse monoclonal anti‐HA (Clone No. C29F4; Cat No. MMS‐101P; Covance, Princeton, NJ, USA), anti‐phospho‐p38 MAPK (Cat No. 4631; Cell Signaling Technology, Boston, MA, USA), anti‐p38 MAPK (Cat No. 8690; Cell Signaling Technology, Boston, MA, USA), anti‐phospho‐Erk1/2 (Cat No. 4377S; Cell Signaling Technology, Boston, MA, USA), and anti‐Erk1/2 (Cat No. 4695S; Cell Signaling Technology, Boston, MA, USA). The primary monoclonal antibody used as a housekeeping control was a monoclonal antibody against β‐actin (Cat No. C1313; Applygen, Beijing, China).