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- Supplemental Fig. 1 - Supplemental Fig. 1 - Supplemental Figure 1. Characterization of the phospho-Thr171 CPEB monoclonal antibody. A, ELISA results were measured at 450 nm absorbance using increasing concentrations of the pCPEB antibody and 50 ng of the phospho-peptide used to generate the monoclonal antibody. The pCPEB antibody bound to the phospho-peptide at increasing concentrations. At all concentrations tested, the pCPEB antibody did not bind the nonphospho-peptide. B, Purified CPEB-GST fusion proteins (4 ?g), WT or Thr171Ala, were phosphorylated in vitro with CaMKII (50 nM) for the indicated time points and then Western blotted with the pCPEB antibody or a total CPEB antibody. pCPEB immunoreactivity was not observed with either WT CPEB without CaMKII phosphorylation or with Thr171A CPEB with CaMKII phosphorylation, demonstrating specificity of the pCPEB antibody. C, Western blots of hippocampal neurons treated with ionomycin (1 ?M) for 5 min and then probed with the pCPEB antibody. Preadsorption of the pCPEB antibody with the phospho-peptide used to generate the antibody eliminated all pCPEB immunoreactivity.