Figure 6

Downregulation of PKC isoforms by specific PKC siRNA. A, BV-2 cells transfected with DY-547 labeled RISC-free siRNA in 24-well plate were photographed using a Nikon Eclipse TE2000-U. The white field shows total cells, TRITC represents positively transfected cells, and merge stands for overlaid images. B, BV-2 cells were transfected with individual PKC isoform-specific siRNAs for 48 hr. Total RNAs were isolated from each transfected group for analysis of gene expression by reverse transcriptase PCR using primer pairs specific for PKC δ, ε, θ, η, α, β and GAPDH. The products were run on a 1% agarose gel impregnated with ethidium bromide. The bands were visualized under UV light. In each transfection panel, starting from the left, the lanes stand for DNA marker, RISC-free control siRNA and specific PKC siRNA, respectively. GAPDH was used as a loading control. C, Total mRNA was extracted from BV-2 cells that were transfected with PKC isoform siRNA or RISC free control siRNA (CON) for 48 hr and subjected to cDNA synthesis. The correspondent cDNA was then used as template for quantitative real time PCR with SYBR Green PCR Master mix. ***, p < 0.001 were obtained when the PKC siRNA transfected groups were compared to the RISC-free siRNA control group. A representative experiment of three that were performed is shown.