Fig. 4. Enrichment of α1FLAGβ3 GABAARs following photolabeling with MQ189, a sulfated NAM neurosteroid PAL, and KK123, a non-sulfated PAM neurosteroid PAL. The alkyne moiety of the covalently attached PAL reagent was coupled to a cleavable biotin-azide linker MQ112 using click chemistry. Photolabeled receptors were then enriched on streptavidin-agarose beads and eluted by cleavage of the linker. The figure shows an anti-FLAG-western demonstrating enrichment of photolabeled GABAARs. Lane 1 lysate of membranes expressing α1FLAGβ3 GABAARs and shows the mass (∼52 kDa) of the α1FLAG subunit. The bands at 52 kDa indicates that both MQ189 (Lane 2) and KK123 (Lane 3) photolabel the α1FLAG subunit of the GABAAR.
