Figure 1.

The effects of SE on the cell surface stability and phosphorylation of GABA_ARs. A, B, SE modulates GABA_A receptor surface expression levels. Hippocampal slices from control (Con) or SE were labeled with NHS-biotin and lysed, and biotinylated proteins were purified on avidin. A, Cell surface and total fractions (1/10 of input; In) were immunoblotted with antibodies against GABA_A receptor α1–2, α4–5, β3, γ2, and δ subunits, GABA_B R1 and R2 subunits, 14-3-3ζ, and actin. B, Data in SE (open bars) were normalized to the cell surface levels evident in control slices (filled bars; 100%). C, SE decreases the phosphorylation of S408/9 in the GABA_AR β3 subunit. Top, Hippocampal slices from control and SE were subjected to biotinylation, and cell surface fractions were blotted with a phospho-specific antibody against S408/9 in the β3 subunit (p-β3) and anti-β3 antibodies (T-β3). Bottom, The ratio p-β3/T-β3 signals were then measured in control (filled bars) and SE (open bars) with values normalized to those in control (100%). In all panels, asterisks indicate significant difference from control (p < 0.01; Student's t test; n = 6–8). D, Hippocampal slices were prepared from Con, and animals were injected with 35 (p35) and 350 mg/kg (p350) pilocarpine, respectively. Slices were biotinylated, and cell surface and total fractions were isolated and immunoblotted with the respective antibodies as indicated.