Validation of GABA_A receptor subunits as novel interaction partners of MPP2.

(a) Bacterially expressed GST-MPP2-SH3GK and GST-PSD-95-SH3GK were incubated with crude brain synaptosome preparations. After GST pull-down, compared to bead control, GABA_AR α1 was efficiently enriched in the GST-MPP2-SH3GK pull-down, as detected by western blot with αGABA_AR α1 and αGST antibodies. (b) GABA_A receptor subunit constructs with an N-terminal pHluorin were acquired from Addgene and expressed in CHL V79 cells together with N-terminally FLAG-tagged MPP2. Pull-down of (i) GABA_AR α1, (ii) GABA_AR α2, and (iii) GABA_AR β3 with Ms αGFP or normal Ms-IgG resulted in co-precipitation of FLAG-MPP2 as detected by western blot with αFLAG-HRP and αGFP antibodies. GABA, γ-aminobutyric acid; MPP2, membrane protein palmitoylated 2; PSD, postsynaptic density.