Figure 1

CD63 glycosylation in breast cancer cells. A) MDA-MB-231-luc-D3H2LN (MM231-LN) (upper panel) and MCF7-ADR cells (lower panel) were transiently transfected with control (N.C.) or RPN2 siRNAs. After 2 days in culture, RPN2 expression was detected using immunoblotting. β-actin was used as a loading control. B) MM231-LN (upper panel) and MCF7-ADR cells (lower panel) were transiently transfected with the N.C. or RPN2 siRNAs. After 2 days in culture, the cell extracts were subjected to qRT-PCR. The values on the y-axis are plotted relative to the expression level of N.C., which is defined as 1. C) MM231-LN (upper panel) and MCF7-ADR cells (lower panel) were transiently transfected with N.C. or RPN2 siRNAs. After 2 days in culture, CD63 expression was detected using immunoblotting. β-actin was used as a loading control. Dashed lines show the difference between glycosylated CD63 in the presence or absence of RPN2 siRNA treatment. D) Whole cell lysates were collected from MM231-LN (upper panel) and MCF7-ADR cells (lower panel) that were transiently transfected with N.C. or RPN2 siRNA and treated with PBS for 6 hours at 37°C. N.C. and RPN2 siRNA-transfected cells were treated with N-glycosidase for 6 hours at 37°C. CD63 glycosylation was detected by immunoblotting. β-actin was used as a loading control. The CD63 molecular weights of 25 (non-glycosylated), 35 (lower-glycosylated) and 50 kDa (higher-glycosylated) are indicated with arrows to the right.