CD63 is required for Syt VII trafficking to lysosomes. (A) HeLa cells were treated with Lipofectamine alone, control siRNA, or CD63 siRNA. Immunoblotting was performed with anti-CD63 antibodies. Antiactin antibodies were used as loading controls. (B) Fluorescence confocal images of cells treated with control or CD63 siRNA after fixation, permeabilization, immunofluorescence with antibodies to CD63, and DNA staining with DAPI. (C and D) HeLa cells treated with control or CD63 siRNA were transduced with adenovirus expressing Syt VII–YFP and Lamp1-RFP or GalT-RFP and imaged by live confocal microscopy. CD63 transcriptional silencing abolished the colocalization of Syt VII–YFP with Lamp1-RFP (C), causing its retention in the Golgi (D). (E) HeLa cells treated with control or CD63 siRNA were transduced with adenovirus expressing Syt VII–YFP, Lamp1-RFP, and CFP-resCD63, encoding silent mutations rendering the transcript resistant to CD63 siRNA-mediated silencing. CFP-resCD63 expression was not inhibited by CD63 siRNA, allowing normal colocalization of Syt VII–YFP with Lamp1-RFP. Bars, 6 µm.