Ecl immunoblotting system

Transfected cells were lysed for 30 min at 4 °C in RIPA buffer (0.15 M NaCl, 0.5% sodium-deoxycholate, 0.1% SDS, 0.05 M Tris-HCl (pH 8.0), 1% NP-40) supplemented with a protease inhibitor. Spinal cords taken from 16-week-old ALS Tg-mice were lysed for 1 h at 4 °C in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH8.0), 0.5% NP-40) containing protease inhibitor. Cell or tissue lysates were quantified with the Bradford assay (Bio-Rad, Hercules, CA, USA). Then, the samples underwent SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and were transferred to nitrocellulose membranes. The membranes were incubated overnight with the indicated primary antibodies at 4 °C. Following incubation with the appropriate secondary antibody for 1 h at room temperature, the proteins were visualized with an enhanced chemiluminescence (ECL) immunoblotting system, as described by the manufacturer (GE Healthcare Life Sciences, Chicago, IL, USA) [53 (link)].

For western blot analysis, hUCB-MSCs were washed with ice-cold 1 × PBS and lysed with RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche). Protein concentrations were determined by the Bradford assay. Lysates were separated using Novex, NuPAGE, and Bolt precast gels (Invitrogen) under denaturing conditions and transferred to nitrocellulose membranes. After blocking with 1% bovine serum albumin (BSA) solution, membranes were immunoblotted with various antibodies and then probed with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence (ECL) immunoblotting system (GE Healthcare).