Fig. 3

Ser24-PP2Ac is phosphorylated upon stimulation in a PKC and IGBP1-depndent manner. A, B Time course of S24-PP2Ac phosphorylation upon TPA stimulation. αT3-1 (A) or PC3 (B) cells were serum starved (0.1% FCS, 16 h), followed by stimulation with TPA (250 nM) for the indicated times. Then, the cells were harvested and subjected to Western blot analysis using an anti-phospho Ser24-PP2Ac raised for this study (see Methods). The lower graphs represent means ± SD of three experiments. Significance of change from non-stimulated (time 0) control is calculated. * p < 0.01, ** p < 0.05. C, D PKC is involved in Ser24-PP2Ac phosphorylation upon stimulation. Serum starved (0.1% FCS, 16 h) αT3-1 (C) or PC3 (D) cells were pretreated with PKC inhibitor GFx (3 µM, 20 min) or DMSO control and then either stimulated with TPA (250 nM, 30 min) or left untreated. Cells were harvested, followed by analysis of the cell extracts by Western Blotting with the indicated Abs. The bar-graphs in the lower panels represent means ± SD of 3 experiments. Significance of change from DMSO, non-stimulated (-) control is calculated. * p < 0.01, ** p < 0.05. E, F IGBP1 is involved in the TPA-induced Ser24-PP2Ac phosphorylation. αT3-1 (E) or PC3 (F) cells, were treated with SiRNA of IGBP1 or SiRNA control, serum starved (0.1% FCS, 16 h) and either stimulated with TPA (250 nM, 30 min) or left untreated. The cells were harvested, and extracts were analyzed with the indicated Abs. The lower bar-graphs are means ± SD of three experiments. Significance of change between non-stimulated SiRNA control (-) and the other stimulated and non-stimulated cells is calculated. * p < 0.01, ** p < 0.05