Fig. 4

Identification of IGBP1 as a key regulator of the PP2A switch. a Si-RNA screen to identify the PP2A B subunit involved in the PP2A switch. αT3-1 cells were transfected with the indicated Si-RNAs by incubation of two 6 cm plates for each Si-RNA (6 h, 20 nM SiRNA pool). Then, the cells were transferred to a fresh medium with 10% FCS, and the cells were left for 24 h recovery followed by serum starvation for 14 h. Next, for each SiRNA, the cells in one plate were stimulated with TPA (250 nM, 15 min) and the other plate was left untreated. Finally, the cells were lysed and the extracts were subjected to Western blotting with the indicated Abs. The MW of the gAKT and pAKT in all lanes is 64 kDa. b SiRNA of IGBP1 reduces the stimulated AKT dephosphorylation in PC3 cells. PC3 cells were transfected with the indicated SiRNA and treated as described for the αT3-1 cells in A. In the final stage, the cells were subjected to Western blotting with additional Abs as indicated. c Combined SiRNAs of the two PP2Aa subunits does not affect AKT dephosphorylation. PC3 cells were transfected with the combined SiRNA of both PP2Aa isoforms or scramble control (Si-CTRL) for 6 h. Forty-eight hr later, the cells were serum-starved (16 h) and then stimulated with TPA (250 nM, 30 min, +) or left untreated (-). Cell extracts of each treatment were then subjected to Western blotting using the indicated Abs. d Stimulated IGBP1 interactions with PI3K’s p85 (PI3K), AKT and PP2Ac. αT3-1 (left panels) or PC3 (right panels) cells were serum starved and stimulated with TPA (250 nM) for the indicated times. The cells were harvested and then IGBP1 was IPed from the cell extracts using anti-IGBP1 Ab. CoIPed proteins as well as the proteins in the loaded extracts were detected using Western blotting with the indicated Abs. e Quantification of the results in d. The graphs represent the average and standard errors of three independent experiments. Significance of change relative to non-stimulated (NT) control is calculated for each protein and cell line. * p < 0.01, ** p < 0.05. f IGBP1 is required for the interaction of PP2A with PI3K and AKT. αT3-1 (left panels) or PC3 (right panels) were transfected with the SiRNA (75 nM) of IGBP1 or Scramble control (CTRL) for 6 h (50 nM SiRNA pool), after which the cells were moved to medium containing 10% FCS for 48 h to recover followed by serum starvation for additional 16 h. Then the cells were stimulated with TPA (250 nM, 30 min), harvested, and PP2A was IPed from the cell extracts using anti-PP2A. The CoIPed proteins were detected using Western blotting with the indicated Abs. g Quantification of the results in E. The results shown represent means ± standard errors of three experiments. Significance of change from non-stimulated (NT) control is calculated for each protein and cell line. * p < 0.01, ** p < 0.05