Anti na k atpase antibody

Transfected HEK293T cells were collected in modified RIPA buffer (50 mM Tris (pH = 7.4), 150 mM NaCl, 1% NP-40, 0.2% sodium deoxycholate, 1 mM EDTA) and 1% protease inhibitor cocktail (Sigma). Collected samples were subjected to gel electrophoresis using 4–12% BisTris NuPAGE precast gels (Invitrogen) and transferred to PVDF-FL membranes (Millipore). Monoclonal anti-α1 subunit antibodies (NeuroMab) and polyclonal anti-γ2 subunit antibodies (Alomone or Millipore) were used to detect GABA_A receptor subunits. Anti-Na+/K + ATPase antibody (Abcam) was used as a loading control. IRDye® (LI-COR Biosciences) conjugated secondary antibody was used at a 1:10,000 dilution in all cases. Membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences). The integrated intensity value of bands was determined using the Odyssey Image Studio software (LI-COR Biosciences).
For protein digestion, cell lysates were incubated with enzyme Endo H or PNGase F in G7 or G5 reaction buffer, respectively (New England BioLabs). Digestion proceeded for 3 h at 37 °C and was stopped with 5% β-mercaptoethanol (Sigma). Treated samples were then subjected to SDS-page electrophoresis and western blot.

Cell-surface biotinylation was carried out as previously reported (3 (link)). Briefly, 350-μm coronal slices were prepared with a vibratome and then immediately transferred to ice-cold artificial cerebrospinal fluid (ACSF) oxygenation with 95% O₂/5% CO₂. The slices were then incubated twice in ice-cold 0.75 mg/ml NHS-SS-biotin (Thermo Scientific) for 45 min. Then, slices were washed several times to stop the excess biotin reaction followed by adding lysis buffer with protease inhibitors. Hi-Capacity NeutrAvidin beads (Thermo Scientific) was used to incubate supernatant for 16–18 h at 4°C to pull down and elute the proteins with elution buffer. The protein concentration was determined, and equal amount protein was subjected to SDS-PAGE analysis as Western blot assay. GABA_A receptor α5 subunit antibody (1:1,000, Millipore, ab9678), β-actin antibody (1:4,000, Santa Cruz Biotechnology, sc-517582), and anti-Na+/K+ ATPase antibody (1:500, Abcam, ab58475) were used for Western blot analysis. The expression of α5 was normalized to its respective loading controls in the surface (NKA) and total (β-actin). The purity of extracted surface protein was determined by probing the presence of β-actin with antibody.