Complete edta free protein inhibitor cocktail

The primary visual cortex of adult mice was dissected under deep anesthesia with sodium pentobarbital (83 mg/kg, i.p.). The bilateral cortices of each mouse were mixed as one sample. Proteins were extracted with RIPA lysis buffer containing 0.01 M PBS (pH 7.4), 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, and complete EDTA-free protein inhibitor cocktail (Roche Applied Science). After blocking in 5 % nonfat dry milk (wt/vol, in 20 mM TBST), the membrane was incubated with rabbit anti-NR2A (1:1000, Abcam, ab77980), rabbit anti-NR2B (1:1000, Cell Signaling Technology, 4212S), rabbit anti-GAD65 (1:2000, Proteintech, 20746-1-AP), mouse anti-GAD67 (1:1000; Abcam, ab26116), and mouse anti-β-actin (1:15,000; Abcam, ab6276) antibodies, followed by the respective HRP-conjugated secondary antibodies (1:2500-1:10,000; Promega). The membrane was then developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Western blot analysis was repeated multiple times, and the optical density of each band was determined using ImageJ software and normalized to that of β-actin.

Preserved cells were thawed on ice for 30 min and resuspended in buffer A (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 7.4) containing cOmplete EDTA-free protein inhibitor cocktail (Roche, 1 tablet/10 mL), lysozyme (1.0 mg/mL) and DNAse (1 U/µL). The suspension was incubated on ice for 30 min and sonicated (6 pulse, each 10 s). Cellular debris was removed by centrifugation at 15,000×g for 30 min at 4 °C. Recombinant, N-and C-terminally His-tagged proteins were purified using a Ni–NTA Superflow cartridge (1 mL, Qiagen) and the Fast Protein Liquid Chromatography (FPLC) system (BioRad). The proteins were eluted with filter sterilized buffer B (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 7.4). Protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific) and purity was checked by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with Coomassie Blue (BioRad) and destained with destaining reagent (10% acetic acid and 20% methanol).