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Fig. 7. Specific reduction of SV2A and SV2C expression by siRNAs. (A) BHK cells were transiently transfected with plasmids encoding SV2A or SV2C. The cells were then collected and the expression of the exogenous proteins was analyzed by western blotting using anti-SV2A (a) or anti-SV2C (b) antibodies. (B) Silencing of the genes was assessed by co-transfecting SV2A or SV2C with either an empty pSilencer vector (control) or a pSilencer vector containing a sequence that directs the synthesis of SV2A siRNA-1, siRNA-2, siRNA-3 (a) or SV2C siRNA-1, siRNA-2, siRNA-3 (b). -, lane with non-transfected cells. After 3 days the cells were homogenized and equal amounts of protein were analyzed by western blotting with anti-SV2A or -SV2C antibodies. (C) INS-1E cells were transiently co-transfected with a plasmid encoding GFP and with an empty vector (control) or with a vector containing either the SV2A siRNA-3 or the SV2C siRNA-2 silencers. Four days later, GFP-expressing cells were enriched by FACS separation. The cells were then homogenized and the same amount of protein was separated by SDS-PAGE and immunoblotted with anti-SV2A (a) or anti-SV2C (c) antibodies. The membranes (a,c) were stripped and then incubated with anti-SV2C (b) and anti-SV2A (d) antibodies, respectively. The lower parts of the membranes were incubated with an antibody against synaptotagmin IX (syt IX). Arrows indicate proteins unrelated to SV2. (D) INS-1E cells were transiently transfected with GFP and with an empty vector (control) or with both SV2A siRNA-3 and SV2C siRNA-2. Half of the homogenates prepared as just described, was loaded on a SDS-PAGE and immunoblotted with anti-SV2A (a) and the other half was immunoblotted with anti-SV2C (b).
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