Figure 5

Syntenin-1-binding deficient mutants of CD63 fail to recover L1-7 reactivity.

(a) Representative pictures of wild-type (wt) and mutant CD63. Rat1 cells were transfected with a plasmid encoding CD63 mutant T7 (human) and stained with species-specific CD63 antibodies, sc-5275 (human) and AD1 (rat). CD63-T7 mutant (green) colocalises with endogenous rat CD63 (red). (b) CD63-T7 mutant cannot interact with syntenin-1. CHO cells were cotransfected with plasmids encoding flag-tagged syntenin-1 and human CD63 as indicated. Immunoprecipitation was carried out using mouse anti-human CD63 mAb 6H1. Precipitated proteins were detected by Western blotting using rabbit Flag (lower panels) or CD63 antibodies (upper panels). Lysates were loaded as positive controls for transfection. Asterisks mark non-specific bands. (c–e) Recovery of L1-7 reactivity using CD63 mutants. CD63 depleted HeLa cells were transfected with control or mutant CD63 plasmid and infected with HPV16. (c) CD63 mutants efficiently reexpress CD63 after respective knockdown. (d) Shows representative pictures of CD63 and L1-7 double staining in different CD63 mutants. (e) L1-7 reactivity was quantified as in Fig. 2e,f. *P < 0.05 significant decrease compared to control, ^$P < 0.05 significant increase compared to cells transfected with CD63 siRNA and control plasmid.