FIG. 4.

Overexpression of Cbl-b induces muscle atrophy in vivo. (A) Mock vector or pCEFL-Cbl-b-HA was injected directly into the rat tibialis anterior muscle. Two weeks later, muscle sections were stained with anti-HA and anti-ubiquitin (Ub) antibodies and counterstained with H&E. Arrows represent injection points, which were indicated by bromophenol blue. Scale bar, 100 μm. (B) Cross-sectional areas of myofibers expressing Cbl-b-HA (from the boxed area of panel A, containing 50 myofibers) were calculated with WinRoof software. We also measured the area of 50 myofibers in uninjected or mock vector-injected myofibers as controls. Data are means ± standard deviations (n = 4). *, P < 0.05 for results for injected myofibers compared to those for control myofibers. (C) Homogenates from muscle blocks expressing Cbl-b or injected with mock vector were immunoprecipitated (IP) with an anti-IRS-1 or anti-HA antibody, followed by immunoblotting (IB) for the indicated proteins. Results shown in this figure are representative of four experiments. MMSTD, molecular mass standards.