Lf qc0101

For brain tissue preparation, mice were deeply anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) administered intraperitoneally. Mice were perfused transcardially with heparin dissolved in PBS (pH 7.2). The dissected brain tissues were frozen at -80 °C for Western blotting. Tissues were homogenized with total RNA and protein isolation kit (NucleoSpin® RNA/Protein #740933.50, Macherey-Nagel, Dűren, Germany). The protein samples were quantified with Pierce™ BCA Protein Assay Kit and 50 μg protein sample were used for each Western blot. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 1,000; Dako #Z0334), anti-Iba1 (rabbit, 1:300; Wako #NB100-1028), anti-EAAT1 (rabbit, 1:1,000; Santacruz # sc-15316), anti-EAAT2 (rabbit, 1:1,000; Cellsignaling #3838), anti-Cx43 (mouse, 1:1,000; Santacruz #sc-59949), anti-PSD95 (mouse, 1:2,000; Thermo #MA1-046), anti-NeuN (mouse, 1:1,000; Millipore #MAB377) anti-GAPDH (rabbit, 1:10,000; Abfrontier #LF-PA0018). Secondary antibodies were conjugated with horse-radish peroxidase (HRP) (1: 10,000, Invitrogen). The HRP signals were visualized using an enhanced chemiluminescent (ECL, Abfrontier #LF-QC0101, Gyeonggi-do, Korea) substrate.

Samples were lysed using ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 0.5% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1% Triton X-100) containing a protease inhibitor cocktail (#535140, Calbiochem, Darmstadt, Germany) and a phosphatase inhibitor cocktail (#P3200-001, GenDEPOT, Baker, TX). After brief sonication, the lysates were centrifuged at 16,000 x g for 30 min at 4°C, and the supernatants were collected. In case of mice tissues (brains and hearts), after lysing and homogenizing the samples with TRIzol (#TR118, Molecular Research Center Inc, Cincinnati, OH, USA), proteins were isolated according to the manufacturer’s protocol. The protein concentrations were determined using the DC Protein Assay Reagents Package (#5000116, Bio-Rad, Hercules, CA). Proteins were resolved by SDS-PAGE, transferred to PVDF or NC membrane, and immunoblotted with the indicated primary antibodies and subsequently with horseradish peroxidase-conjugated secondary antibody (#G-21040, Invitrogen or #111-035-003, Jackson ImmunoResearch, West Grove, PA). Proteins were then visualized using an enhanced chemiluminescence (ECL) system (#LF-QC0101, AbFrontier, Seoul, Korea). The band intensities were determined using ImageJ (NIH, Bethesda, MD).