Anti rest

The lysates of tissues or cells were homogenized in RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 4% protease inhibitor). The following primary antibodies were used: anti-TRPV4 (1:500; Alomone Labs), anti-KCa3.1 (1:500; Abcam), anti-α-SMA (1:1000; Sigma), anti-phospho-JNK/c-Jun antibodies (1:1000, Cell Signaling Technology, Danvers, MA, United States), anti-REST (1:500; Santa Cruz, Santa Cruz, CA, United States), and anti-β-actin (1:1000; Santa Cruz). HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (1:3000; Amersham Biosciences) were used for 1 h at room temperature. Bands were quantified by ImageJ software and normalized to the β-actin band as loading control (Schneider et al., 2012 (link)).

The following antibodies and chemicals were purchased: anti- Cullin-4B, anti-FLAG (M2), and anti-α-tubulin antibodies, mouse IgG-agarose, monoclonal anti-HA-agarose, and anti-FLAG M2 affinity gel from Sigma-Aldrich Co. (St. Louis, MO); anti-caspase-3, anti-caspase-9, anti-cleaved caspase-3, and anti-cleaved caspase-7 antibodies from Cell Signaling Technology (Beverly, MA); anti-Apaf-1 antibody from Millipore (Temecula, CA) and Abcam (Cambridge, United Kingdom); anti-multi ubiquitin antibody (FK2) from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan); anti-p62 (C-terminal) antibody (specific to amino acids 421–440 of human p62 protein) from PROGEN Biotechnik GmbH (Heidelberg, Germany); anti-HA (HA.11 and Y-11) and anti-myc antibodies from Covance (Richmond, CA) and Santa Cruz Biotechnology (Santa Cruz, CA); and anti-ATF4, anti-REST, and anti-Bcl-xS/L antibodies from Santa Cruz Biotechnology. HRP-conjugated anti-rat, anti-mouse, anti-rabbit, and anti-guinea pig IgG (H + L) antibodies were purchased from Southern Biotech (Birmingham, AL); MG132 from Calbiochem (La Jolla, CA); etoposide, E64d, pepstatin A, and cycloheximide from Sigma-Aldrich Co. All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), Kanto Chemical (Tokyo, Japan), and Sigma-Aldrich Co.

While under deep anesthesia (5% sevoflurane), the L4-L5 segments of spinal cord were isolated quickly and stored in -80 ºC. Tissue samples were homogenized in lysis buffer and centrifuged at 13,000 rpm for 10 min at 4 °C. Supernatant was removed. Bradford method was used to determine the protein concentration. Samples (70 μg) were separated on SDS-PAGE (6%) and transferred onto a nitrocellulose membrane. The filter membranes were blocked with 5% nonfat milk for 1 h at RT (room temperature) and incubated with the primary anti NR2B (rabbit affinity purified polyclonal antibody; 1:1000, Abcam, Hong Kong, China), anti REST (rabbit affinity purified polyclonal antibody; 1:300, Santa Cruz Biotechnology, Santa Cruz, CA) or anti β-actin (mouse affinity purified monoclonal antibody; 1:1000, ZSGB-BIO, Beijing, China). The membrane was washed with TBST buffer and incubated for 1h with the secondary antibody conjugated with horseradish peroxidase (1:5000) for 1 h at RT and visualized in ECL solution followed by film exposure. The density of specific bands was quantified with a computer-assisted imaging analysis system (IPLab software, Scanalytics, Fairfax, VA).