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Fig. 6 | Cell Communication and Signaling

Fig. 6

From: GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch

Fig. 6

Interaction of IGBP1 with PP2Aa. a, b IGBP1 interacts with PP2Aa with and without stimulation. Serum-starved αT3-1 a or PC3 b cells were treated with TPA (250 nM) for the indicated times. Then, anti IGBP1 Ab or non-relevant Ab (non-relevant IgG, Con) were used for IP, and the CoIPed proteins were detected with PP2Aa Ab. The amount of IPed IGBP as well as PP2A and IGBP loading were detected using with the indicated Abs. c Quantification of the results in a, b. The graphs represent means ± standard errors of three experiments for the indicated proteins.* p < 0.01. d PLA verifies PP2Aa interaction with IGBP1. αT3-1 (left) and PC3 (right) cells were cultured on cover slips. The cells were then serum starved, and either stimulated with TPA (250 nM, 15 min) or left untreated (both panels of αT3-1 cells and TPA 0 in PC3 cells) followed by fixation. Protein–protein interactions of PP2Aa and IGBP1 were detected using the PLA kit with their cognate Abs as described under Experimental Procedures. The control in the αT3-1 cells means that no PP2Aa Ab was used in the reaction. e Non-denaturing gel electrophoresis to detect PP2A complexes. Cells were grown to 70% confluence, starved for 16 h, and then were either stimulated with TPA (250 nM, 30 min; +) or left untreated (-). The formation of PP2A complexes and whether they contain PP2Ac (c), PP2Aa (A), and IGBP1 (I) was detected by non-denaturing gels, followed by blotting with the indicated Abs. The lower molecular weight proteins are not presented here. The molecular weight standard is shown here although the migration of the marker proteins in this native gel does not reflect the actual mass as it depends on the charge of each protein as well. Thus the position of the markers provides just a rough estimation of the actual molecular weight. f PP2Aa does not affect AKT interaction with IGBP1. The same extracts from Fig. 4C were taken for additional experiments and subjected to IP with anti IGBP1 Ab. The CoIPed and IPed proteins and loading extracts were detected using Western blotting with the indicated Abs

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