Western Blot Analysis of GABAA Receptors

The HEK293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM of Tris (pH = 7.4), 150 mM of NaCl, 1% NP-40, 0.2% sodium deoxycholate, 1 mM of EDTA) and 1% protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). The collected samples were subjected to gel electrophoresis using 4–12% BisTris NuPAGE precast gels (Invitrogen) and transferred to polyvinylidene difluoride fluorescence-based (PVDF-FL) membranes (Millipore, Burlington, MA, USA). The primary antibodies used to detect GABA_A receptors were as follows: Mouse α1 subunit antibody (1:500; NeuroMab, 75–136), rabbit β3 subunit antibody (1:500; Novus, NB300-199), and rabbit γ2 subunit antibody (1:500; Millipore, AB5559). The Mouse anti-Na+/K+ ATPase antibody (1:000; DSHB, a6F) was used to determine the density of the loading control. IRDye^® (LI-COR Biosciences, Lincoln, NE, USA) conjugated secondary antibody was used at a 1:10 000 dilution in all cases. The blotted membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences). The band-of-interest integrated intensity values were determined using the Odyssey Image Studio software (LI-COR Biosciences). Biotinylation protocols were described previously [23 (link)].