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. 2016 Mar 1;7(16):21601–21617. doi: 10.18632/oncotarget.7802

Figure 5. APIP is an essential activator of HRG-β1/ERBB3 in gastric cancer cells.

Figure 5

A. APIP-interacting proteins were purified from SNU-16 cells expressing 3xFLAG-tagged APIP by co-immunoprecipitation assay using FLAG M2 affinity gel. The bound proteins were resolved by SDS-PAGE and prepared for LC-MS/MS analysis. CBB, coomassie brilliant blue. B. Inhibition of HRG-β1-dependent ERBB3 phosphorylation and its downstream signals by APIP knockdown. Serum-starved SNU-16 control and APIP knockdown cells were treated with 10% FBS, 2 μM Insulin, 50 ng/ml IGF, 100 ng/ml EGF, 50 ng/ml HRG-β1 or 50 ng/ml FGF2 for 10 min and subjected to western blotting. C. Analysis of phospho-ERBB3 (Y1289) in a panel of gastric cancer cell lines by western blotting.D. Inhibition of ERBB3 activity by APIP knockdown in complete medium. Whole-cell extracts of SNU-16 control and APIP knockdown cells were analyzed with western blotting. E. APIP overexpression sensitizes SNU-620 cells to HRG-β1. SNU-620 control and APIP-overexpressing cells were stimulated with 2 ng/ml HRG-β1 and subjected to western blotting. Starv., serum starved. F. and G. ERBB3 knockdown inhibits cell growth and suppresses AKT and ERK1/2 activity. Cell growth (middle) and death rates (bottom) of SNU-16 control cells (shControl) or ERBB3 knockdown cells (shERBB3 #1 and #2) were assessed and analyzed by western blotting (top). The results represent mean ± S.D. (n = 3). (F). Whole cell lysates were examined by western blotting (G). H. Enhanced HRG-β1 signaling in APIP transgenic MEFs. WT and APIP transgenic MEFs were treated with 10 ng/ml HRG-β1 and harvested for immunoprecipitation (IP) assay. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01.