Pugnac 100um

Mouse brain tissues were dissected after perfusion of animal and snap frozen and lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS, complete protease inhibitors 2X; Roche, and PUGNAc 100uM; Sigma). Tissue lysates were mixed with 4x NuPage LDS loading buffer (Invitrogen), loaded on a 4%–12% SDS polyacrylamide gradient gel (Invitrogen), and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in Tris-Buffered Saline with Tween (TBST) and incubated with mouse anti-O-GlcNAc antibody (RL2) ab2739 (1:500, Abcam), rabbit anti-OGT antibody (H-300) sc-32921 (1:1000, SC Biotech), rabbit anti-PSD-95 antibody #2507 (1:300; CS Technology), rabbit anti-NR2B ab65783 (1:1000, Abcam), rabbit anti-synapsin-1 ab18814 (1:1000, Abcam), mouse anti-synaptophysin (SY38) MAB5258 (1:1000, EMD Millipore), and mouse anti-GAPDH (6C5) ab8245 (1:10,000, Abcam). Horseradish peroxidase-conjugated secondary antibodies and an ECL kit (Clarity ECL, Bio-Rad) were used to detect protein signals. Multiple exposures were taken to select images within the dynamic range of the film. Selected films were scanned (300 dpi) and quantified using FIJI software (Version 1.0). GAPDH bands were used for normalization.