Figure 2.
GABAAR β3-subunit-specific phosphorylation in response to BDNF. a, b, Immunoprecipitation with control rabbit IgG, or antibodies specific for the β3-, β2-, or γ2-subunit of GABAARs was performed using lysates from either [32P]orthophosphate-labeled cultured cortical neurons incubated in the absence (-) or presence (+) of BDNF (100 ng/ml) for 10 min (a), or [35S]methionine-labeled cultured cortical neurons (b). Arrows indicate migration of individual GABAAR subunits. c, Anti-phospho-β3 antibody binds specifically to P-GSTβ3 (lane 3, 100 ng; lane 4, 200 ng) with no cross-reactivity to mock P-GSTβ3 (lane 1, 100 ng; lane 2, 200 ng). d, Equal protein amounts of SDS lysates from mock-transfected COS-7 cells (lane 1), cells transfected with either GABAAR α1- and γ2s-subunits only (lane 2), or in combination with GABAAR β3 wild-type (wt) subunit (lane 3), or β3 mutants containing S408A mutation (lane 4), S409A mutation (lane 5), or S408/9A mutation (lane 6), which were incubated in the absence (-), or presence (+) of forskolin (20 μm) for 20 min, were analyzed by immunoblotting with anti-P-β3 antibody (top), or β3-specific antibody (bottom). Arrows indicate the migration of P-β3 antibody-reactive band detected in lysates containing wt β3-subunit, which was enhanced by forskolin (P-β3, top) or migration of wt or mutant β3-subunits detected by β3-specific antibody (β3, bottom). e, Immunoblot analysis using anti-P-β3 antibody alone (lane 1) or using the same concentration of anti-P-β3 antibody preincubated with either synthetic dephospho-S408/409 peptide (lane 2), or chemically phosphorylated phospho-S408/409 (lane 3), phospho-S408 (lane 4), or phospho-S409 peptide (lane 5) of SDS lysates obtained from cultured cortical neurons incubated in the absence (-) or presence (+) of BDNF (100 ng/ml) for 5 min. All peptides were incubated at 500-fold molar excess. Arrow indicates a migration of a ∼58 kDa band corresponding to phosphorylated neuronal GABAAR-β3-subunit.