Sin3a

Total protein extracts from brain were harvested as described above. Equal amounts of 1 mg of protein were incubated for 1 h at 4°C with 4 μg antibody. Immunoprecipitation was carried out by using protein A beads or protein G beads (Dynabeads; Invitrogen) overnight at 4°C. The immune complexes were washed three times with Hunt buffer. Samples were used for an HDAC activity assay, or they were heated in SDS sample buffer and used for immunoblotting. Primary antibodies used for coimmunoprecipitation were Sin3A (catalog number sc-994X; Santa Cruz), CoREST (catalog number 07-455; Millipore), and MTA1 (catalog number sc-9446; Santa Cruz) antibodies.

Cells were lysed in RIPA buffer, 50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% (v/v) NP40, 0.5% (v/v) Na-deoxycholate, 0.1% (v/v) SDS, and 1 tablet/10 ml Complete, Mini, EDTA-free protease inhibitors (Roche). SDS–PAGE and immunoblots were carried out using standard protocols.
Antibodies were used as follows: SINHCAF [1 (link)], HIF-2α (PA1-16510, Thermo Scientific; 7096, Cell Signaling), β-actin (3700, Cell Signaling), HIF-1α (610958, BD Biosciences), HIF-1β (3718, Cell Signaling), PHD2 (Bethyl A300-322A; 4835, Cell Signaling); p52 (05-361, Merck/Millipore), E2F1 (3742, Cell Signaling), SP1 (07-645, Merck/Millipore), SP3 (sc-644, Santa Cruz Biotech), Sin3A (sc-994, Santa Cruz Biotech; 8056, Cell Signaling); HDAC1 (17-10199; Merck/Millipore), AcH3 (acetylated histone H3; 06-599, Merck/Millipore), p62/STQM (610832, BD Biosciences), poly-ADP ribose polymerase (PARP) (9532, Cell Signaling), cleaved PARP (9541, Cell Signaling), caspase-3 (9662, Cell Signaling), cleaved caspase-3 (9661, Cell Signaling), and LC3B (3868, Cell Signaling).

Dissected brains were immediately frozen in liquid nitrogen and stored at −80°C. For protein extraction, frozen brains were manually homogenized in Hunt buffer (20 mM Tris-HCl [pH 8.0], 100 mM sodium chloride, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche) and phenylmethylsulfonyl fluoride (PMSF) in a glass homogenizer. After full-speed centrifugation, the supernatant containing the soluble protein fraction was further used. Equal amounts of 20 to 30 μg of proteins were separated by SDS-PAGE (10% gels) and transferred onto nitrocellulose membranes (Protran; Whatman) according to standard protocols. For detection, the Enhanced Chemiluminescence kit (PerkinElmer) was used. HDAC activity assays were performed with brain protein extracts as previously described (9 (link)). Primary antibodies for immunoblotting were HDAC1 (10E2 or Sat13), HDAC2 (3F3), Sin3A (catalog number sc-994; Santa Cruz), CoREST (catalog number 07-455; Millipore), MTA1 (catalog number sc-9446; Santa Cruz), PKCδ (catalog number 610397; BD), and β-actin (catalog number A5316; Sigma) antibodies.