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![Identification of the phosphorylation site of Par3 by ERK2. A, The direct phosphorylation of Par3 by ERK2. Purified MBP or MBP-Par3 deletion mutants were incubated with recombinant ERK2 in the presence of [γ-32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. B, The phosphorylation of Par3 point mutants by ERK2. Purified MBP, MBP-Par3-4N-WT -S1116A, -T1145A, -S1316A, or -T1328A was incubated with recombinant ERK2 in the presence of [γ-32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. C, The alignment of ERK2 phosphorylation sites in Par3 homologs (rat, mouse, human). D, The phosphorylation of Par3-WT or -S1116A in COS7 cells. COS7 cells were transfected with EGFP-Par3-WT or -S1116A and cultured for 24 h. The cells were serum-starved for 24 h and then treated with or without 1 μm OA for 2 h. Cell lysates were incubated with anti-GFP antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-phospho-MAPK/CDK substrates and anti-GFP antibodies.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5157/pmc06705157/pmc06705157__zns9991342990002.jpg)
Journal: The Journal of Neuroscience
Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization
doi: 10.1523/JNEUROSCI.4210-12.2013
Figure Lengend Snippet: Identification of the phosphorylation site of Par3 by ERK2. A, The direct phosphorylation of Par3 by ERK2. Purified MBP or MBP-Par3 deletion mutants were incubated with recombinant ERK2 in the presence of [γ-32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. B, The phosphorylation of Par3 point mutants by ERK2. Purified MBP, MBP-Par3-4N-WT -S1116A, -T1145A, -S1316A, or -T1328A was incubated with recombinant ERK2 in the presence of [γ-32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. C, The alignment of ERK2 phosphorylation sites in Par3 homologs (rat, mouse, human). D, The phosphorylation of Par3-WT or -S1116A in COS7 cells. COS7 cells were transfected with EGFP-Par3-WT or -S1116A and cultured for 24 h. The cells were serum-starved for 24 h and then treated with or without 1 μm OA for 2 h. Cell lysates were incubated with anti-GFP antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-phospho-MAPK/CDK substrates and anti-GFP antibodies.
Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST ( Nishimura et al., 2004 ); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit
Techniques: Purification, Incubation, Recombinant, In Vitro, SDS Page, Silver Staining, Autoradiography, Transfection, Cell Culture, Western Blot