Anti hdac3 antibody

For HDAC3 qChIP, DP thymocytes were enriched using the EasySep Mouse Streptavidin RapidSpheres Isolation kit (#19860, StemCell Technologies) to remove SP and DN thymocytes with biotin-conjugated anti-CCR7 (4B12), anti-IL-7Rα (A7R34), anti-H2K (AF6-88.5), anti-CD44 (IM7), and anti-CD25 (PC61.5) as well as anti-TCRγδ (UC7-13D5) antibodies. For SP thymocyte enrichment, Mouse PE Positive Selection kit (#18554, StemCell Technologies) was used positively select for CCR7⁺ thymocytes (PE-conjugated anti-CCR7 (4B12)). ChIP was performed according to Pchelintsev et al. (2016) (link), with the following adjustments: After cell lysis and brief sonication (four mins, 30 s on/30 s off; Bioruptor Pico (Diagenode, Inc.)), equal volume of 2x MNase buffer (35 mM Tris-HCl pH 7.5, 25 mM NaCl, 120 mM KCl, 2 mM CaCl₂) was added to each sonication sample along with 3.75 U/μl of MNase (Cell Signaling Technologies #10011S) at 37°C for 15 mins. Anti-HDAC3 antibody (#85057, Cell Signaling Technologies) was used to isolate chromatin bound to HDAC3 after chromatin fragment size was confirmed to be less than 500 bp by gel electrophoresis. Isolated DNA was used to perform real-time PCR. Graphs depict fold enrichment over regions without HDAC3 binding (Rpl30 primers, Cell Signaling Technologies (#7015)). See Supplementary file 1 for primer sequences.

Proteins in BMDMs were extracted by RIPA lysis buffer (Thermo Scientific) containing protease or phosphatase inhibitors. The proteins (50–70 µg) were separated by 10% SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The PVDF membrane was blocked in 5% nonfat milk (Sigma, USA) for 1 h at room temperature and then incubated with primary antibodies [anti-HDAC3 antibody (#85,057), anti-STAT3 antibody (#9139), anti-p-STAT3 antibody (#9131), and anti-β-actin antibody (#3700) (all antibodies from Cell Signaling Technology, dilutions 1:1000)] at 4℃ overnight. The secondary antibodies conjugated to horseradish peroxidase were incubated for 1 h at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence system (Amersham Biosciences), according to the manufacturer’s instructions.