Fig. 5.

GPC3 activates ERK phosphorylation. (A) ERK phosphorylation in GPC3-expressing NIH3T3 stable lines. Cells were serum starved for 24 h. Forty micrograms of cell extracts were analyzed by western blot with either anti-phospho-ERK or anti-ERK antibody. Whole-cell extracts from 12-O-tetradecanoylphorbol 13-acetate-treated HEK293 cells (lane 2) were used as a positive control. (B) ERK phosphorylation in GPC3-knocked-down HuH-7 cells. shRNA was transiently transfected into HuH-7 cells. After serum starvation, 35 μg cell extracts were subjected for western blot analysis with anti-phospho-ERK, anti-ERK, 1G12 or anti-actin. ERK phosphorylation decreased after GPC3 knockdown. (C) ERK phosphorylation in PLC-PRF-5 cells. Cells were stably transfected with p gpc3-GFP (GPC3) or control vector (vector) and serum starved for 24 h. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (D) ERK phosphorylation in HA22T/VGH cells. Cells were stably transfected with pcDNA-gpc3 [wild-type GPC3 (WT-GPC3)], RR → AA, P25-29A or control vector (vector) and serum starved at 0.5% fetal calf serum for 24 h. Cell extracts were immunobloted with either anti-phospho-ERK, anti-ERK or 1G12. (E) IGF-II knockdown by siRNA (siIGF-II). Chemically synthesized, double-stranded siRNAs for IGF-II and control were transfected into HEK293 cells. Total RNA was extracted and analyzed by reverse transcription–polymerase chain reaction. Specific primers for IGF-II were used in polymerase chain reaction and actin was served as the RNA loading control. (F) ERK phosphorylation was decreased by IGF-II knockdown. HuH-7 cells were transfected with either control or IGF-II siRNA and serum starved. Cell extracts were analyzed with either anti-phospho-ERK or anti-ERK. (G) Growth rate of HuH-7 cells after IGF-II knockdown. Cells were seeded in 12-well plates in triplicate, transfected with either control or IGF-II siRNA and were serum starved. Cells were harvested at 48 h intervals.