Dynamic effects of ALP inhibition via 3-MA or BFA on p62 protein levels and a surrogate UPS substrate in cultured neonatal rat ventricular myocytes (NRVMs).
Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu, Ad-RFP) 48 hr before 3-methyladenine (3-MA) or bafilomycin A1 (BFA) treatment. A ∼ C, The time course of protein level changes in the GFPu/RFP ratio and the p62/β-tubulin ratio in NRVMs treated with 3-MA (3 mM) or vehicle control. Representative images of western blots (A) and quantitative data from 3 repeats (B, C) are shown. Paired t-test, *p<0.01. D and E, 3-MA and BFA concentration-dependently increases GFPu/RFP ratio at 24 hours. At 24 hr after the drug treatment, the cells were harvested for total protein extraction and western blot analyses of the indicated proteins. Representative images (D) and pooled densitometry data (E) are shown. Two-way ANOVA followed by the Scheffé's test; *p<0.05, **p<0.01 vs. CTL; n = 3 repeats/group.