Trans blot cell transfer system

Whole cell lysates were collected using NP-40 lysis buffer (Invitrogen, FNN0021) with protease and phosphatase inhibitor cocktail (Thermo Scientific, 78443). 50μg total protein was separated by SDS–PAGE using Any kD™ Mini-protean TGX precast polyacrylamide gels (BioRad, 4569033), and transferred to PVDF membranes using the Trans-Blot Cell Transfer System (BioRad). Membranes were incubated overnight with antibodies against TBP (Cell Signaling, 85155, 1:1000), EZH1 (Abcam, ab64850, 1:1000) at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 hour at room temperature. Chemiluminescence was detected using SuperSignal West Pico Plus Chemiluminescent substrate (Thermo Scientific, 34579).

Ipsilateral and contralateral lumbar spinal segments (including L4 and L5 segments) of SNL and sham-operated rats were separated and homogenized for immunoblotting. The tissues were lysed in ice-cold RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.5 mg/ml BSA). After centrifugation of the homogenates, the protein concentration was determined by using a detergent-compatible protein assay with a bovine serum albumin standard. Samples were separated on a 7.5% (w/v) sodium dodecyl sulfatepolyacrylamide gel and transferred onto a nitrocellulose membrane (Amersham, Pittsburgh, PA) with a Trans-Blot Transfer Cell system (Bio-Rad, Hercules, CA). Membranes were incubated with the indicated primary antibody overnight at 4°C, and immunoreactivity was detected by enhanced chemiluminescence (ECL, Amersham). We used antibodies against Cx43 (1:8000, Sigma-Aldrich, St. Louis, MO, Cat. #: C6219), Cx36 (1:1000, Life technologies, Grand Island, NY, Cat. #: 51-6200), glial fibrillary acidic protein (GFAP; 1:2000, Millipore, Billerica, MA, Cat. # MAB360), and β-actin (1:10,000, Chemicon, Temecula, CA). We then quantified the intensity of immunoreactive bands of interest on the autoradiograms using Image J 1.46r software (NIH, Bethesda, MD). Actin staining was used as an internal control for protein loading.