Fig. 4.

Control of GluR1 Ser845 phosphorylation by cotransfection with cDNA encoding Cα-PKA and PKI. A,Top panel, Representative immunoblot showing reduction in GluR1 Ser845 phosphorylation with cotransfection of a cDNA encoding the peptide inhibitor PKI. The same immunoblot was probed with an antibody that recognizes phosphorylated Ser845, and stripped and reprobed using an antibody against GluR1. P/C is the ratio of S845-P signal intensity normalized to GluR1 C-terminal antibody signal intensity (total GluR1) for PKI or PKA treatment versus control and illustrates the reduction in basal phosphorylation by the PKI vector (p < 0.05). For comparison, a P/C ratio of 1.0 is shown for the control lane (R1).Bottom panel, Immunoblot showing that cotransfection with PKI also reduces Ser831 phosphorylation (p < 0.05). B, Top panel, Immunoblot showing enhancement of GluR1 Ser845 phosphorylation with cotransfection of cDNA encoding Cα-PKA. An antibody selective for phosporylated-Ser845 (S845-P) was used to probe the blot, and the same blot subsequently was stripped and reprobed with a GluR1-selective antibody (see Materials and Methods). The bar graph to the right shows the quantification of the Cα-PKA-induced enhancement of Ser845 phosphorylation as the ratio of immunodetected phosphorylated GluR1 to total GluR1; error bars are SEM (p < 0.05; paired t test). Control bars (R1) were set at 1.0 for comparison. The level of basal phosphorylation inA and B are similar and appear different because of the different exposure times for the ECF analysis.Bottom panel, Immunoblot and analysis showing the lack of effect on phosphorylation of GluR1 Ser831 by cotransfection with Cα-PKA.