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. 2013 Aug 14;33(33):13270–13285. doi: 10.1523/JNEUROSCI.4210-12.2013

Figure 3.

Figure 3.

Phosphorylation of Par3 by ERK2 in vivo. A, Specificity of the phosphospecific anti-phospho-Par3 (pS1116) antibody. MBP-Par3-4N-WT (1 pmol) containing the indicated amounts of Par3-4N-WT or -S1116A phosphorylated by ERK2 was subjected to SDS-PAGE. Immunoblot analyses with anti-pS1116 (top) and anti-MBP antibodies (bottom) were performed. B, The phosphorylation of EGFP-Par3 in COS7 cells. COS7 cells were transfected with EGFP-Par3-WT and treated with DMSO, 50 μm PD98059, or 20 μm U0126 for 4 h. The cell lysates were analyzed by immunoblotting with anti-pS1116 and anti-GFP antibodies. The phosphorylation of ERK1/2 was also examined with anti-phospho-ERK1/2 antibody. Data represent the mean ± SEM of three independent experiments. **p < 0.01 (Dunnett's multiple-comparison test). C, D, The phosphorylation of endogenous Par3 in hippocampal neurons at 3 DIV. C, Hippocampal neurons were treated with DMSO, 50 μm PD98059, or 20 μm U0126 for 4 h and then with or without 1 μm OA for 2 h. The cell lysates were analyzed by immunoblotting with anti-pS1116 and anti-Par3 antibodies. The phosphorylation of ERK1/2 was also examined with anti-phospho-ERK1/2 antibody. Data represent the mean ± SEM of three independent experiments. Asterisks indicate statistical significance. *p < 0.05 (Tukey's multiple-comparison test). **p < 0.01 (Tukey's multiple-comparison test). ***p < 0.001 (Tukey's multiple-comparison test). D, The cells were treated with DMSO or 20 μm U0126 for 4 h and then with or without NT-3 (100 ng/ml) for 30 min. The cell lysates were analyzed by immunoblotting with anti-pS1116 and anti-Par3 antibodies. The phosphorylation of ERK1/2 was also examined with anti-phospho-ERK1/2 antibody. Data represent the mean ± SEM of four independent experiments. *p < 0.05 (Tukey's multiple-comparison test). **p < 0.01 (Tukey's multiple-comparison test). ***p < 0.001 (Tukey's multiple-comparison test). E, The spatial distribution of phosphorylated Par3 at Ser-1116 in hippocampal neurons. Hippocampal neurons were transfected with Myc-Par3-WT or -S1116A at 2 DIV. Neurons were fixed at 3 DIV and then immunostained with anti-Myc and anti-pS1116 antibodies. A merged image and the ratio (phosphorylated Par3 to total Par3) are shown. Scale bars, 20 μm. The graph plots the fluorescence intensities of Par3 phosphorylated at Ser-1116 (green) and total Par3 (red) along the line (from X to Y).