CAV1 is ubiquitinated. (A) HEK293 cells were transfected with CAV1-HA or CAV1-K*R-HA, and CAV1 was immunoprecipitated using anti-CAV1 (N20) antibody. Probing blots with anti-ubiquitin (FK2) antibody revealed ubiquitinated CAV1 species in immunoprecipitates prepared from cells expressing wild-type CAV1-HA but not from cells expressing the lysine-null mutant CAV1-K*R-HA. Migration of molecular mass standards is indicated in kilodaltons. (B) Pulse-chase analysis showed that turnover of CAV1-K*R-HA was slowed down by 47% when compared with wild-type CAV1 (CAV1-HA). Error bars indicate mean ± SD from three samples collected on two experimental days. (C) Ubiquitin staining accumulated and colocalized with overexpressed CAV1-HA in Rab5-positive EE of CV1 cells. (D–F) Accumulation of ubiquitin in EE was not observed in cells transfected with the lysine-null mutant CAV1-K*R-HA (D), in untransfected cells (E), or in cells transfected with mRFP-Rab5/EGFP-Rab7 (F). Single confocal sections of fixed cells are shown. (C–F, bottom) Insets show enlargements of boxed areas. Bars, 10 µm.