FIG. 1.

CD63 knockdown in MDM. Seven days after monocyte isolation (day 0), MDM were nucleofected with scrambled or CD63-specific siRNAs. CD63 knockdown was assessed by immunofluorescence and by Western blot analysis 2, 4, and 6 days after nucleofection. (A) Permeabilized cells were stained with anti-CD63, followed by Alexa Fluor 594 anti-mouse and Hoechst dye for immunofluorescence analysis. Scale bars = 10 μm. (B) For Western blot analysis, 2 × 10⁶ MDM were solubilized in lysis buffer. Cell proteins were separated in nonreducing buffer on 10% SDS-polyacrylamide gel electrophoresis gels and immunoblotted for CD63 and clathrin heavy chain (CHC). CD63 runs as a broad smear due to glycosylation (18). The blots were quantified using Bio-Rad Quantity One software, and band intensities were normalized against clathrin. The loss of CD63 is expressed as a percentage of the CD63 levels in control cells (bottom right in the blots).