Akt2-dependent activation of RalA in mouse gastrocnemius muscle as detected by immunofluorescent microscopy of isolated gastrocnemius muscle fibers.

(a) The expression vector for Myr-p110α, together with either one of two siRNA duplexes against mouse Akt2 (#1 and #2) and a mixture of NC siRNA duplexes, was introduced into gastrocnemius muscle of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous RalA and Akt2 were visualized by immunofluorescent staining with anti-RalA and anti-Akt2 antibodies, respectively. Myr-p110α was visualized by immunofluorescent staining with an anti-HA antibody. Activated RalA (RalA·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after treatment with GST-V5^×3-Sec5(1–99). Scale bar, 20 μm. (b) Activation of RalA shown in (a) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001.