Figure 2.

Kinases present in brain lysate phosphorylate GABA_AR α2 at Ser-359. A, GST alone or GST fused to the intracellular loop of the α2 subunit (WT or S359A) was labeled with 10 μCi of ³²P and incubated with brain lysate as a source of kinase for 7 min at 30 °C. Fusion proteins were then subjected to SDS-PAGE and visualized with autoradiography (left). Right, quantification of ³²P incorporation. Data are means ± S.E. (error bars). **, p < 0.01; ****, p < 0.0001; one-way ANOVA, Tukey's post hoc test, n = 3. B, phosphopeptide map of GST-α2 WT and S359A. ³²P-Labeled α2 bands from A were excised, trypsinized, blotted on TLC plates, and subjected to electrophoresis followed by ascending chromatography. Colored circles represent the two major tryptic peptides found in GST-α2 WT. The red circle highlights a tryptic peptide in GST-α2 WT that is missing in S359A. The blue circle highlights a tryptic peptide in GST-α2 WT that is still present in S359A.