Fig. 2.

Dox enhances GFPu degradation in cultured ventricular myocytes. GFPu was forced to express in cultured neonatal rat ventricular myocytes (NRVMs) through adenoviral gene delivery. A: direct fluorescence micrographs of GFPu adenoviruses (Ad-GFPu)-infected NRVMs and control virus (Ad-β-Gal)-infected NRVMs. Scale bar = 10 μm. B: representative image of immunoblot (IB) analysis for GFPu in the immunoprecipitates (IP) of an anti-ubiquitin (Ub) antibody. C: Dox (10 μM) reduced GFPu protein levels in NRVMs. The full-length native GFPu band is indicated by an arrow (the same for D). D: GFPu and a mutant desmin (Des) were coexpressed in NRVMs. Compared with the treatment of the vehicle control (mock), Dox (10 μM for 24 h) significantly decreased the level of GFPu proteins [both the native form (indicated by the arrow) and the ubiquitinated forms, which have higher molecular weights] but not desmin protein. E: bar graph summarizing 4 independent repeats of the GFPu and desmin protein denisitometry data of the experiments shown in D. AU, arbitrary units. *P < 0.05 vs. mock. F: representative image of Western blot analysis for GFPdgn in the cycloheximide (CHX) chase experiment. Cardiomyocytes were isolated from adult mice expressing GFPdgn (a shorter form of GFPu) and cultured for 24 h before being treated with Dox or Dox + CHX. G: summary of the densitometry data from 2 repeats of the duplicated CHX chase experiments shown in F. *P < 0.05 vs. 0 h; #P < 0.05 and ##P < 0.01 vs. Dox.