Figure 3. Rho signaling-mediated activation of ezrin facilitates nuclear accumulation of β-DG.

A. Treatment of C2C12 cells with the toxin C3, a specific Rho inhibitor, decreased β-DG nuclear accumulation. Lysates from cells treated or not with C3 were analyzed by SDS/Western blotting using an antibody that recognizes phosphorylated ezrin, raxidin and moesin (p-ERM). The membranes were reprobed with antibody for total ezrin (upper panel). Cells grown on coverslips were treated without or with C3, fixed and immunolabeled using anti-β-DG primary and a fluorescein-conjugated secondary antibodies and nuclei stained with DAPI. Cells were imaged and quantitative analysis for Fn/c of β-DG was performed as described in Figure 2 (bottom right panel) showing significant differences between cells treated or not with C3. B. Nuclear and cytoplasmic fractions from control and C3-treated cells were analyzed by SDS/Western blotting using anti- β-DG antibodies (upper panels). Immunodetection of Sp3 and Calnexin (Clnx) was used as loading control for nuclear and cytoplasmic fractions respectively. The n/c ratio for β-DG was quantified and plotted as per Figure 2 (bottom panel) with significant differences determined by p value. C. C2C12 cells treated with LPA (inductor of the Rho pathway) show increased β-DG nuclear accumulation. Lysates from cells pretreated with LPA or PBS for 0–30 min were analyzed by SDS/Western blotting using anti-p-ERM or anti-ezrin antibodies (upper left panel). Control and LPA-treated cells were immunolabeled for β-DG and imaged as before (left panel) with significant differences between control and treated cells. D. Cytoplasmic and nuclear extracts of untreated and LPA-treated cells were analyzed by SDS/Western blotting using anti-β-DG antibodies. Sp3 and Calnexin were employed as loading controls (upper panels). The n/c ratio for β-DG shows significant differences between control and treated cells. All results representing the mean +/- SD from three separate experiments were analyzed by Student t-test.