Figure 3

NHERF1 potentiates ERM phosphorylation in LPA-stimulated cells. (a, b) Effect of NHERF1 depletion on LPA-induced ERM phosphorylation. OVCAR-3 cells were transfected with siRNA duplexes specific for NHERF1, NHERF2 or control siRNA (luciferase GL3). One day after transfection, the cells were re-plated onto collagen-coated culture dishes. One day later, the cells were serum-deprived for 24 h and treated with 1 μM LPA for various times (a) or with various doses of LPA for 10 min (b) as indicated. LPA-induced levels of cpERM, pAKT, pSGKα/β and pERK in the cell lysates were determined by western blot analysis with the indicated phospho-specific antibodies (a, b). The amounts of total ERM proteins, NHERF1 and NHERF2 in the same cell lysates were also determined using the appropriate cognate antibodies (a). Black arrows indicate cognate target proteins, and the blue arrowhead indicates nonspecific bands in the western blot image (a). These results are representative of three independent experiments. The relative intensity of each immunoblot image is shown in the graph at the right side. Data represent mean ±s.e.m. *P<0.05 and ^**P<0.01, t-test. (c) Expression levels of NHERF1 and ERM proteins in ovarian cell lines derived from either normal or cancerous epithelium. NHERF1 and ERM protein levels in the cell lysates of HIO-80, SK-OV-3 and OVCAR-3 are shown together with the GAPDH level as an input control. (d, e) Potentiation of ERM phosphorylation by overexpression of NHERF1. HIO-80 cells were transfected with GFP-NHERF1 (WT) or its C-terminally deleted form (ΔCT). Immunoblot analyses with anti-GFP and anti-NHERF1 antibody were performed to determine the expression level of each NHERF1 construct and the degree of overexpression of GFP-NHERF1 relative to the endogenous NHERF1 level, respectively (d). LPA-induced levels of cpERM and pERK, as well as total ERM level, were determined as described above. All of these results represent three independent experiments.