Figure 2.

Effects of rottlerin on PKCδ and PKCα enzyme activity and protein levels. (A) PKCδ and (B) PKCα kinase activities. The indicated cell lines were treated with rottlerin (20 µM) for 48 h, then harvested for analysis. 400 µg of protein lysates were used for immunoprecipitation by isozyme-specific antibodies, and the immunopurified proteins were assayed using an artificial substrate. (C) PKCδ activity measured by in vitro assay. PKCδ was pulled down from cell lysates by IP and treated with rottlerin (20 µM) for 4 h, and then subjected to an in vitro kinase assay. (D) PKCδ protein levels in cells after rottlerin treatment. 40 µg aliquots of the same protein lysates used in the studies in panels A and B were separated and immunoblotted using an anti-PKCδ monoclonal antibody. Ratio: refers to the value of PKCδ expression/β-actin expression measured by Software BandLead 3.0. (E) PKC isozyme expression levels after treatment with rottlerin. 1×10⁵ cells were plated in each well of a 6-well plate. Cells were treated with rottlerin (20 µM) or with the same volume of vehicle after they reached 80% confluence. After 48 h, cells were harvested, lysed in 50 µl lysis buffer and the lysates separated and subjected to immunoblot analysis using anti-PKC isoform-specific antibodies. Lanes 1 to 4 represent: Balb, KBalb, NIH/3T3 and NIH/3T3-Ras cells respectively. The immunoblots shown in the left and right panels were separated on the same gel and immunoblotted at the same time. Because the samples in the left panel came from untreated cells, whereas the samples in the right panel came from rottlerin-treated cells, and because rottlerin-treatment inhibited cell growth/proliferation, fewer numbers of cells were obtained in these groups, and therefore less protein/lysate was available for assay from the treated group compared to the vehicle control group. The mean activities ±SD were obtained from at least three independent experiments, and assays from immunoprecipitations using an anti-β-actin antibody were used to determine the background activity, which was subtracted. Student’s t-test was used for statistical analysis. (*, p <0.01; ♦, p <0.001).