Figure 6

Knockdown of ErGPCR by RNAi blocks 20E-induced nuclear translocation and phosphorylation of Calponin in the HaEpi cells. (A) Green fluorescence represents Calponin detected with anti-Calponin antibody. Blue fluorescence indicates DAPI-stained nuclei. The image in the white box shows the efficacy of ErGPCR knockdown (by dsRNA incubation for 24 h followed by 1 μM 20E induction for 1 h), determined by RT-PCR; N, C, and dsG indicate ErGPCR transcripts in normal cells, dsGFP-treated cells, and dsErGPCR-treated cells, respectively. (B) 20E-induced phosphorylation of Calponin was detected with anti-Calponin antibody by western blot analysis. Cal-P indicates phosphorylated Calponin. (C) The effect of anisomycin on 20E-induced phosphorylation of Calponin was analyzed using anti-His monoclonal antibody by western blot analysis. Cal-GFP (56 kDa) is the fused expressed Calponin with GFP in the HaEpi cells; Cal-GFP-P indicates phosphorylated Calponin-GFP; GFP (35 kDa) was expressed alone in the cells as a control. The cells were incubated with 10 μM anisomycin for 1 h and then treated with DMSO or 1 μM 20E for 1 h.