Ripa lysis buffer

RIPA lysis buffer (Zsbio) was used to extract protein from tissues and indicated cells. BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) was used to measure protein concentration. A total of 50 μg of protein was separated on 10% SDS-PAGE and blotted onto 0.22-μm nitrocellulose membranes. The membranes were blocked for 2 h with 5% nonfat dry milk diluted with tris-buffered saline (TBS) and incubated with primary antibodies [rabbit polyclonal anti-REST (1:100), rabbit polyclonal anti-MMP9 (1:100; Abcam), and mouse monoclonal anti-β-GAPDH (1:2,000; Abcam)] overnight at 4°C. The membranes were washed with TBS containing 0.1% Tween 20 (TBST) and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:2,000; goat anti-mouse, 1:2,000; Santa Cruz, Santa Cruz, CA, USA) for 1 h at 37°C. Enhanced chemiluminescence reagent (Merck Millipore, Darmstadt, Germany) was used to detect the signal on the membrane. The data were analyzed via densitometry using the Image-Pro plus software 6.0 (Bio-Rad, Hercules, CA, USA) and normalized to the expression of the internal control (GAPDH).