Increased proteasome assembly is a delayed response to muscle denervation. (A) Innervated and denervated muscle homogenates were analyzed by native polyacrylamide gel electrophoresis and in-gel LLVY-AMC (β5 proteasome substrate) hydrolysis. The native gel was then photographed and visualized under UV light to detect the activities of proteasome holoenzyme complexes (RP2-CP, RP1-CP); 20S activity was detected by the addition of SDS (panel b). The native gel was then subjected to immunoblotting for PSMC1(Rpt2) and PSMD2 (Rpn1) in native (panels c and d) and denaturing (panel e) gels. (B) shGankyrin downregulates gankyrin in NIH-3T3 cells. mRNA preparations from cells transfected with shLacz control or shGankyrin were analyzed by RT-PCR and specific primers to gankyrin. Means ± SEM are presented as a ratio to shLacz. N = 3 wells of cells per shRNA. *, P < 0.05 versus shLacz by one-tailed paired Student’s t test. (C) Gankyrin is required for proteasome assembly in atrophying mouse muscles. Innervated and denervated TA muscles expressing shGankyrin or scrambled shLacz (control) were analyzed by native gel and in-gel LLVY-AMC hydrolysis assay. (D) Downregulation of gankyrin in denervated muscles attenuates fiber atrophy. Measurements of cross-sectional areas of 590 fibers expressing shGankyrin (green fibers, also express GFP) and adjacent 590 non-transfected fibers. N = 5 mice. Statistics in Table 1. A representative image of electroporated muscle is shown. Fiber membrane staining (red) using wheat germ agglutinin (WGA). Scale bar: 50 μm. Source data are available for this figure: SourceData F3.