Original western blot images

Description

Original western blot images

Steps to reproduce

Western bolt analysis was performed as we described previously.23,24 Tissues from vmPFC were lysed in 100 mM HEPES containing 200 mM NaCl, 10% glycerol, 2 mM Na4P2O7, 2 mM DTT, 1 mM EDTA, 1 mM benzamidine, 0.1 mM Na3VO4, 1 μM pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 10 μM PMSF at PH 7.4. After lysis for 30 min in ice, the sample was centrifuged at 12,000 g, 4 °C for 15 min and the supernatant was collected. The samples containing equivalent amounts of protein were applied to acrylamide denaturing gels (SDS-PAGE). The separated proteins were transferred onto Immobilon®-P Transfer Membranes (Millipore, Burlington, MA, USA). Blotting membranes were incubated with blocking solution [7.5% nonfat dried milk powder dissolved in PBST buffer (pH 7.5, 10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20)] for 1 h at room temperature, washed three times, and then were incubated with primary antibodies in PBST overnight at 4 °C. After several washes with PBST buffer, the membranes were incubated for 2 h with appropriate horseradish peroxidase-linked secondary antibody. Then the membranes were processed with enhanced chemiluminescence (Millipore, Cat #WBKLS0500). The films were scanned with ChemiDOCTM MP Imaging System (Bio-Rad), avoiding densitometric saturation, and densitometry was performed using Image Lab software (BioRad). Primary antibodies used were rabbit anti-BEST1 (1:2000; Cohesion, Cat# CPA9240), rabbit anti-GluA1(1:2000; Abcam, Cat# ab109450, RRID: AB_10860361), rabbit anti-GluA2 (1:2000; Abcam, Cat# ab133477, RRID: AB_2620181), rabbit anti-nNOS (1:2000; Thermo Fisher Scientific, Cat# 61-7000, RRID: AB_2313734), rabbit anti-stargazin (1:1000; Millipore, Cat# 07-577, RRID: AB_310726), rabbit anti-β-actin (1:6000; Bioss, Cat# bs-0061R, RRID: AB_10855480), rabbit anti-β-tubulin (1:6000; Bioworld Technology, Cat# BS1482M) and mouse anti-GAPDH (1:6000, Kangcheng, Cat# KC-5G4, RRID: AB_2493106). Secondary antibody used was goat anti-rabbit HRP (1:12,000; Multi Science, Cat# GAR0072) and goat anti-mouse HRP (1:15,000, Multi Science, Cat# GAM0072, RRID: AB_2827834).

Institutions

• Nanjing Medical University

  Jiangsu, Nanjing

Categories

Licence