Fig. 3

PP2Ac is detached from PI3K and interacts with AKT upon stimulation. a, b Increased AKT-PP2Ac interaction upon stimulation. Serum starved αT3-1 (a) and PC3 (b) cells were stimulated with TPA (250 nM) for the indicated times and harvested. Then, PP2A was IPed using anti-PP2A and the CoIPed AKT was detected using the indicated Abs. The graphs in the bottom panels represent means ± standard errors of three experiments. * p < 0.01, ** p < 0.05. c, d PP2Ac interacts with PI3K’s p85 in resting cells and the interaction is decreased upon stimulation. Serum starved αT3-1 (c) and PC3 (d) cells were stimulated with TPA (250 nM) for the indicated times and harvested. Then, PP2A was IPed using anti-PP2A and the CoIPed p85 was detected using the indicated Abs. The graphs in the bottom panels represent means ± standard errors of three experiments. * p < 0.01, ** p < 0.05. e, f Using PLA to follow PP2Ac interaction with AKT and PI3K’s p85. αT3-1 (left) and PC3 (right) cells were cultured on cover slips. The cells were then serum starved, stimulated with TPA (250 nM, 30 min) and fixed. Protein–protein interactions were detected and quantified using the PLA kit, as described in experimental procedures. The bar graphs in the lower panels represent means ± standard errors of a representative experiment that was reproduced 3 times. Significance of change from non-stimulated (NT) is calculated. * p < 0.01, ** p < 0.05