Fig. 1.

EGF induces a tensin3/cten switch that leads to RhoA activation via DLC1. (A) Endogenous DLC1 binds to tensin3 and cten in MCF10A cells. Tensin3 and cten were respectively immunoprecipitated from cell lysate and immunoblotted for DLC1 (Top). IgG, nonspecific IgG control. The blot was stripped and reprobed for cten (Middle) or tensin3 (Bottom). (B) The effect of EGF treatment on the protein levels of DLC1, cten, and tensin3 and on the activation of RhoA, ERK, and EGFR. Serum-starved MCF10A cells were incubated for 12 h with EGF (10 ng/mL). Immunoblots (IB) of the whole cell lysates (WCL) were used to assess the protein levels. Rhotekin-conjugated beads were used to pull down activated RhoA, followed by IB with anti-RhoA. ERK activation was examined using an antibody specific for phosphorylated ERK (p-ERK). EGFR activation was assessed by IB with 4G10 (anti-pTyr). (C) Colocalization of DLC1 with tensin3 or cten in MCF10A cells before or after EGF treatment. Confocal images were taken for serum-starved MCF10A cells before (-EGF) or after 24 h (+EGF) of EGF treatment. (D and E) Confocal immunofluorescence analysis of RhoA-GTP (D) and total RhoA (E) in serum-starved MCF10A cells before or after EGF treatment. (Scale bars: 10 μm.) Images shown are representative of at least three independent experiments.