Figure 6
PKCδ is involved in facilitating T315 and ILK knockdown-mediated MUC1 phosphorylation and degradation. (a) Effects of shRNA-mediated selective depletion of PKC isoforms α, β, γ, δ and ɛ (two different shRNAs each) on T315-mediated MUC1-C downregulation. Selective PKC isoform-depleted cells were treated with T315 at the indicated concentrations for 24 h. (b) Co-IP analysis of the effect of shRNA-mediated ILK knockdown (left) and T315 (2 μM) treatment for 24 h (right) on MUC1-C phosphorylation and binding with PKCδ in AsPC-1 cells. PKCα was used as a negative control. Equal amounts of cell protein lysates were immunoprecipitated with MUC1-C antibody and protein A/G agarose beads followed by immunoblotting with p-Ser/Thr, PKCα, PKCδ and MUC1-C antibodies. (c) ILK negatively regulates PKCδ activation. Effects of shRNA-mediated knockdown (left) and ectopic expression of ILK (right) on PKCδ and/or PKCα phosphorylation. AsPC-1 cells were transiently transfected with Flag-WT-ILK for 48 h (right). Quantification was done by using ImageJ. Double bands shown in ILK and Flag were quantified separately and summed up together to get fold change compared to the control group. Data shown are representative of at least three independent experiments.
