CAV1 degradation requires endosomal acidification. (A) Degradation time course of endogenous CAV1 and transiently transfected CAV1-HA in CV1 cells as determined by a pulse-chase experiment (see Materials and methods). t_1/2 of CAV1-HA was 13.6 h, as determined by an exponential fit to data points from n = 3 independent experiments. Endogenous CAV1 was degraded much slower (t_1/2 >36 h). (B) Degradation of CAV1-HA was inhibited by 0.2 µM BafA and 20 mM NH₄Cl, which neutralize endosomal acidification, and by 10 µM MG132, an inhibitor of the ubiquitin–proteasome system. Error bars indicate mean ± SEM (n = 3–5 independent experiments). (C) Treatment of HeLa-CAV1-mRFP cells with BafA, NH₄Cl, or MG132 caused accumulation of CAV1-mRFP in endosomal organelles. Single confocal sections of living cells, focus set to perinuclear endosomes. Insets show enlargements of boxed areas. Bars, 10 µm.