Fig. 4

 PKC directly interacts with PP2Ac. Determining PP2Ac-PKC interaction using CoIP. A, B αT3-1 (A) or PC3 (B) cells were transfected with HA-PP2Ac (in PCDNA3) in serum starvation medium (0.1% FCS, 16 h). The cells were then stimulated with TPA (250 nM) for the indicated times, harvested, and PP2Ac was IPed using anti-HA Ab. The CoIPed PKC, as well as the IPed HA-PP2Ac were detected by Western blotting with the relevant Abs. IgG was used as a CoIP negative control. The graphs in the lower panels represent means ± SD of 3 experiments. Significance of change from non-stimulated control (time 0) is calculated. * p < 0.01, ** p < 0.05. C. Proximity ligation assay (PLA) verifies PP2Aa interaction with PKC. αT3-1 (upper panel) and PC3 (lower panel) cells were cultured on cover slips, and were transfected with HA-PP2Ac (in PCDNA3) in serum starvation medium (0.1% FCS, 16 h). The cells were then either stimulated with TPA (250 nM, 5 or 15 min) or left untreated (both panels of αT3-1 cells and TPA 0 in PC3 cells) followed by fixation. Interactions of HA-PP2Ac and PKC were detected using PLA with their cognate Abs as described under Methods. The bar-graphs in the lower panels represent means ± SD of 3 experiments. Significance of change from non-stimulated control (NT) is calculated. * p < 0.01, ** p < 0.05