HSP25 is modified and colocalizes with desmin in subcellular aggregates of the MHCsTNF myocardium. (A) Immunofluorescent data from ventricular cryosections of 3-mo-old WT (d–f) and MHCsTNF (a–c) littermates were double labeled for desmin (a and d; green) and HSP25 (c and f; red) and analyzed by confocal microscopy. Nuclei were stained with DAPI. (B) Protein expression levels of HSP25 in 3-mo-old WT and MHCsTNF mice. (C) Western blot analysis of HSP25 from 2D gel electrophoresis of hearts from WT and MHCsTNF mice. Total protein extracts were electrophoretically analyzed in the first dimension by using pH 4–7 IPGphor strips and in the second dimension by 12% SDS-PAGE followed by immunoblotting analysis using antibody for murine HSP25. The arrow indicates the acidic isovariant that is increased in the MHCsTNF myocardium, and the arrowhead shows the alkaline isovariant that is present only in the sample from the MHCsTNF heart. Bars, 20 μm.