Anti hdac2

Spinal cord sections were prepared from intact and TBI mice. Mice were perfused transcardially with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Spinal cords were dissected, postfixed in the same fixative, immersed overnight in PBS containing 30% sucrose, and then embedded in Tissue-Tek OCT and frozen at −80 °C until use. Sections were prepared using a cryostat (20 µm thickness) and mounted on Matsunami adhesive-coated slides (Matsunami, Osaka, Japan). Cryostat sections were incubated with blocking solution containing 5% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with primary antibodies (anti-PKCγ, Santa Cruz; anti-GFAP, Sigma-Aldrich; anti-Iba1, Wako, anti-NeuN, Millipore; anti-tdTomato, SICGEN; anti-GFP, Thermo Fischer Scientific; anti-HDAC2, Abcam; anti-HDAC1, Abcam; anti-Chx10, Exalpha biologicals; anti-Olig2, Immuno-Biological Laboratories) at 4 °C. Immunoreactivity was visualized using Alexa Flour 488- or 568-conjugated secondary antibodies (Thermo Fischer Scientific). Coverslips were then placed on the slides with mounting medium (Dako). Nuclei were stained using 4′, 6-diamidino-2-phenylindole (DAPI). Images were captured using a laser scanning confocal microscope (FV-1200, Olympus) or a fluorescence microscope (IX83, Olympus).

All drugs were prepared just before use: propofol (Diprivan; AstraZeneca UK limited, Italy: jc393, 20 mL: 200 mg); 20% intralipid (2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC, Houston, TX, USA). HGN antisense was synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Jiangsu province, China).
Anti‐β‐actin and anti‐rabbit IgG secondary antibody were obtained from Cell Signaling Technology (Cell Signaling Tech, MA, USA). Anti‐CREB (Phospho S133), anti‐NMDAR2B, anti‐HDAC2, antisynaptophysin, anti‐Ac‐H4K12 and anti‐Ac‐H3K14 antibodies were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti‐HGN antibody was synthesized by Kitgen Bio‐tech Co., Ltd.(Zhejiang province, China).

The following antibodies were used: anti-PAX8 (GeneTex, GTX101583), anti-PARP (#9532, Cell Signaling), anti-cleaved PARP (#5625, Cell Signaling), anti-GAPDH (#8884, Cell Signaling), anti-Paxillin (ab32084, Abcam), anti-HDAC1 (sc-81598, Santa Cruz), anti-HDAC2 (ab32117, Abcam), anti-HDAC3 (#3949, Cell Signaling), anti-H3K27Ac (ab4729, Abcam), anti-GSDME (ab215191, Abcam), anti-cyclin D1 (ab134175, Abcam). Alexa Fluor 594 phalloidin (A12381) was from ThermoFisher Scientific. Recombinant human FGF18 (#4082–25) was from Biovision. FGF18 ELISA kit (LS-F23007) was from Lifespan. CytoTox 96 Non-Radioactve Cytotoxicity Assay Kit (G1780) was from Promega. In vivo grade D-luciferin (P1042) was purchased from Promega. All inhibitors were bought from Selleck Chemicals. For in vitro assays, inhibitors were reconstituted in DMSO (Sigma-Aldrich) at a stock concentration of 10 mM.

Protein was extracted from the fresh ACP surgical specimens by lysing cells with protease inhibitor cocktail (Roche, USA). After measuring the protein content with a Bradford assay, 20 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and incubated overnight with the following antibodies: anti-HAT, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8 (1:1000, Abcam, USA), anti-CBX4, anti-Runx2, anti-histone (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Proteintech, USA). The specimens were then incubated with secondary antibodies, IRDye800-conjugated anti-rabbit IgG and IRDye680-conjugated anti-mouse IgG (1:15,000, LiCor, USA), for 1 h at room temperature to label the primary antibody. An Odyssey Infrared Image System (LiCor, USA) was used to analyze signal intensities. The densitometry results were first normalized to the density obtained for β-actin or histone and then compared with that of the control to obtain relative fold changes.

The following antibodies were used in the study: anti-Strep Tag (Sigma, SAB2702216), anti-FLAG (Sigma, F1804), anti-V5 (Invitrogen, MA5-15253), anti-IRF3 (Cell Signaling, 4302S), anti-HDAC2 (Abcam, ab124974), anti-Tubulin (Sigma, T5326), anti-STAT1 (Cell Signaling, 14994), anti-STAT1-P (Cell Signaling, 9167), anti-IRF3-S386-P (Abcam, ab76493), and anti-IRF3-S396-P (Cell Signaling, 4947S). Human IFNβ was from Peprotech (300-02BC). The HDAC2 inhibitor Santacruzamat A was from Selleckchem.com (S7595). The HDAC2 siRNA was purchased from Santa Cruz Biotechnology (sc-29345). Lipofectamine RNAiMAX (Invitrogen) was used for the siRNA transfections, which were performed according to the manufacturer’s instructions. 3×FLAG-NSP5 was expressed from the pCDH-CMV-MCS-EF1-Puro vector. The expression plasmids for 2×Strep tagged NSP5, NSP9, NSP10, and EGFP were purchased from Addgene. FLAG-IRF3 and IFNβ-Luc plasmids were gifts from Katherine A. Fitzgerald (UMass Medical School) and Zhijian Chen (UT Southwestern), respectively. The ISRE-Luc reporter vector and the HA-MeV-V expression plasmid were provided by Takeshi Saito (University of Southern California) and Michaela Gack (Cleveland Clinic Florida Research & Innovation Center), respectively.