Fig. 2. Effects of IC86430 on GFPdgn expression and the assembly and activity of 26S proteasomes in mouse hearts.

Adult GFPdgn tg mice were treated with IC86430 (IC; 3 mg/kg, ip; n = 6) or equivalent amount of vehicle (n = 3). Ventricular myocardium was sampled 6 hours after injection for the indicated assays. (A and B) Western blot analysis images (A) and pooled densitometry data (B) for the indicated proteins. (C) Representative fluorescence confocal micrographs of IC86430- or vehicle-treated GFPdgn mouse ventricular myocardium. Scale bars, 50 μm. (D and E) RT-PCR image (D) and densitometry data (E) of myocardial GFPdgn mRNA levels. Duplex RT-PCR for GFPdgn and GAPDH were performed. (F to H) Analyses for the activity and abundance of 26S proteasomes after native gel electrophoresis. Myocardial crude protein extracts were subject to native gel electrophoresis and then in-gel peptidase activity assays using Suc-LLVY-AMC as the fluorogenic substrate; the fractionated proteins in the native gel were then transferred to a polyvinylidene difluoride membrane and subjected to immunoblotting (IB) for Psmb5 to assess the abundance of the doubly capped 26S proteasomes (DC) and singly capped 26S proteasomes (SC). Shown are representative images of the in-gel peptidase activity assay (F, top) and corresponding Western blot (F, bottom), as well as the pooled densitometry data (G and H) of respective assays. All P values in this figure were derived from two-tailed unpaired t test with Welch’s correction. AU, arbitrary units.