FIGURE 8.
USP1 phosphorylation is induced by 20E regulation via PLCG1-triggered calcium signaling. A, Western blot assay for the detection of 20E-induced USP1-His phosphorylation in HaEpi cells using an antibody against the His tag. USP1-P, phosphorylated USP1. The cells were transfected with pIEx-USP1-His plasmids (2.5 μg/ml) and treated with 20E at different concentrations (0.1–5 μm 20E for 1 h) and different times (0.25–24 h with 1 μm). Bar graphs indicate the mean ratios of the abundance of phosphorylated USP1 (USP1-P) to that of nonphosphorylated USP1 quantified by Quantity One (Bio-Rad). B, USP1-overexpressing cells were induced with 1 μm 20E for 1 h, and the protein was extracted and incubated with λ protein phosphatase (λPPase) (5 μm) for 30 min. The PKC inhibitor CC (5 μm) was added before induction with 20E to determine the PKC-mediated USP1 phosphorylation. C, effects of inhibitors and PLCG1 on 20E-induced USP1 phosphorylation. Panel a, 50 μm suramin, 10 μm U73122, 3 μm XeC, 50 μm FL, and 10 μm Pyr3 were used to pretreat the cells for 30 min before 20E induction (1 μm for 1 h). Panel b, PLCG1 depletion by RNAi and overexpression of PLCG1-His or PLCG1ΔSH2-His. D, silencing of DopEcR, ErGPCR, and Gαq by dsRNAs transfection determining 20E-induced USP1 phosphorylation. E, PMA (50 ng/ml) or ionomycin (5 μm) was used to treat cells for 1 h to mimic 20E induction for USP1 phosphorylation. The asterisk indicates the significant difference (*, p < 0.05) statistically analyzed by Student's t test based on three independent replicate experiments.