Erbb3

Protein was extracted from cells using 1X RIPA buffer containing a Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Cell lysate proteins (30 μg) were separated on 4–12% NuPAGE Bis-Tris precast gel electrophoresis and transferred to iBlot 2 polyvinyl difluoride membranes (Invitrogen, Carlsbad, CA). The blots were incubated with the appropriate antibodies to detect the protein level of interest, and the immune complexes were visualized by GE Healthcare Amersham ECL WB detection system (ThermoFisher Scientific). Western blots were probed with antibodies against phosphor-ERBB2/HER-2 (Tyr1248) (Cell Signaling Technologies, Danvers, MA; CST# 2247, diluted 1:800), ERBB2 (CST# 2165, diluted 1:800), phosphor-ERBB3 (Tyr1197) (CST# 4561, diluted 1:800), ERBB3 (CST# 4754, diluted 1:800), phosphor-ERK1/2 (CST# 4370, diluted 1:1000), phosphor-p44/p42 MAPK (diluted 1:1000), total p44/42 MAPK, (diluted 1:1000), total ERK1/2 (CST# 9102, diluted 1:1000), phosphor-AKT S473 (CST# 9271, diluted 1:800), AKT (CST# 9272, diluted 1:800), phosphor-EGFR (CST# 3777, diluted 1:800), phosphor-p90RSK (CST# 11989, diluted 1:800), and Actin (Sigma, St. Louis, MO, diluted 1:10,000).

Proteins were extracted from HO8910 and A2780 cells cultured for 30 min in a 37°C atmosphere containing 5% CO₂. The extracted proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and electrophoretically transferred onto nitrocellulose membranes (Millipore, USA). Standard WB analyses were performed using antibodies specific for CTGF (Santa Cruz, CA, USA), CYR61 (Santa Cruz, USA), FN1 (Sigma), HMGA2 (Sigma), ASAP3 (Sigma, USA), ERBB3 (Sigma, USA), IL-6 (Sigma, USA), IL-1 (Sigma, USA), JUN (Invitrogen, USA), MAP2K8 (Sigma, USA), MMP13 (Sigma, USA), NPNT (Invitrogen, USA), ODC1 (Invitrogen, USA), VEGFC (Sigma, USA) and GAPDH (Santa Cruz, USA). Immunoreactions were visualized using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Santa Cruz, CA, USA) and detected by enhanced chemiluminescence. BandScan software was used to analyze the grayscale values.