Figure 4.
Effect of PKC‐ι siRNA on RWPE‐1 cells and DU‐145 cells. (a) Subconfluent cells (2.5 × 105) were treated with PKC‐ι siRNA complex (50 nm for RWPE‐1 and 150 nm for DU‐145) from 24 to 72 h as described in the Materials and methods section. At indicated times, viable cells were counted using trypan blue exclusion assay and haematocyometry. Three independent experiments were performed and mean number of viable cells and SD were plotted. There is a lower cell viability in PKC‐ι siRNA‐treated cells compared to control siRNA. (b) The same population of treated cells was immunobloted for PKC‐ι, as described in the Materials and methods section. Protein (15 µg) was loaded on each lane for each time point, separated by SDS‐PAGE and Western blotted with anti‐PKC‐ι mouse monoclonal or anti‐PKC‐ζ rabbit polyclonal antibody. There was no or very little PKC‐ι present in PKC‐ι siRNA‐treated cells compared to control siRNA. PKC‐ι siRNA has very little ot no effect on PKC‐ζ indicating specificity. Immunobloting with goat polyclonal anti‐β‐actin shows that the loading of protein is equal in all lanes.