CAV1 ubiquitination and ESCRT machinery are needed for ILV targeting. (A) In CV1 cells expressing mEGFP-mCherry (tandem)–tagged CAV1, mCherry fluorescence did not overlap with mEGFP fluorescence in many endosomal structures, indicating acid-dependent quenching of mEGFP in LE/LYS (Fig. 5 A). (B and C) Treatment of cells with 20 mM NH₄Cl (B) or 10 µM MG132 (C) restored mEGFP fluorescence, which is consistent with neutralized lumenal pH and defects in ILV targeting, respectively. HeLa cells pretreated for 48 h with control siRNA (D) or siRNAs targeting ESCRT components Hrs and Tsg101 (E; double knockdown) were transfected with CAV1-tandem for 12 h and viewed live by confocal microscopy. The presence of yellow endosomes upon knockdown of Hrs and Tsg101 showed involvement of ESCRT machinery in ILV targeting of CAV1. (F) Western blot analysis indicated successful knockdown of Hrs and Tsg101. (A–E) Single confocal sections of live cells are shown. (bottom) Insets show enlargements of boxed areas. Bars, 10 µm.