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. 2016 May 10;11(5):e0155292. doi: 10.1371/journal.pone.0155292

Fig 3. FLJ00068-dependent activation of Rac1 in mouse gastrocnemius muscle fibers.

Fig 3

(A) The expression vector for FLJ68ΔN was introduced into gastrocnemius muscle fibers of wild-type mice. Endogenous Rac1 was detected by immunofluorescent staining with an anti-Rac1 antibody. FLJ68ΔN was detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3 or GST-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (B) Expression vectors for Myr-p110α and Myr-Akt2, together with either one of two siRNA duplexes against mouse FLJ00068 (#1 and #2) and a mixture of NC siRNA duplexes, were introduced into gastrocnemius muscle fibers of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and FLJ00068 were detected by immunofluorescent staining with anti-Rac1 and anti-FLJ00068 antibodies, respectively. Myr-p110α and Myr-Akt2 were detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (C) Activation of Rac1 shown in (B) 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.