FIGURE 7.

Cardiomyocyte free and polymerized tubulin in mice overexpressing PP1. Microtubule network density and free and polymerized β-tubulin in cardiomyocytes isolated from control mice, from mice having cardiac-restricted overexpression of PP1, and the same isolated cells infected for 48 h with AdI-1, an adenovirus encoding PP1 I-1, the endogenous inhibitor of PP1. The greater microtubule network density and concentration of polymerized tubulin in the PP2A mice is returned to control by PP1 I-1 expression. The antibody used for the confocal micrographs and tubulin immunoblots was a monoclonal anti-β-tubulin antibody (clone DM-1B; Abcam). For the immunoblot loading control, a monoclonal anti-GAPDH antibody (clone 6C5; Upstate Biotech) was used in the same samples as those used for free tubulin. For the confocal micrographs, the mean pixel intensity (white level) within the boundary of each cardiomyocyte is given numerically; each micrograph is a single 0.1-μm confocal section taken at the level of the nuclei. For this and two other immunoblots, the densitometric ratio of PP1/control signals was 1.08 ± 0.05 for free tubulin and 2.04 ± 0.09 for polymerized tubulin; for PP1 + I-1/control, it was 1.08 ± 0.03 for free tubulin and 0.92 ± 0.05 for polymerized tubulin. Scale bar, 20 μm.