Anti hdac1

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).

The method for Immunohistochemistry was adapted as described before^{49 (link),50 (link)} briefly, 16 human and 36 mice corneal specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3-µm-thick sections. The sections were then stained using immunohistochemical methods after deparaffinization and rehydration (phosphate-buffered saline (PBS) pH 7.4). Antigen retrieval was performed by immersing sections of tissue in citrate buffer (pH 6.0) for 15 minutes and blocked with 5% normal goat serum for 30 minutes. Specimens were washed three times with PBS after each step. Sections were incubated with anti-histone H3 (Abcam, Cambridge, MA), anti-acetylated histone H3 (Abcam, Cambridge, MA), anti-HDAC1 (abcam, Cambridge, MA), anti-TNFα (Santa Cruz Biotechnology, CA) and TLR4 (Abcam, Cambridge, MA), then followed by application of biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and streptavidin peroxidase. The immunoreactivity was visualized using a 3-amino-9-ethylcarbazole (AEC) kit (Zymed, South San Francisco, CA). Isotype-matched primary antibody was used as a negative control. Slides were counterstained with H&E staining and mounted with a glycerin gelatin mounting medium.

The mouse monoclonal anti-Myc, anti-GFP, and anti-B-actin antibodies were purchased from Sigma (St. Louis, MO). The mouse polyclonal anti-TLE1 and the anti-HDAC1 antibodies were obtained from Abcam (Cambridge, MA). The anti-E-cadherin antibodies Cat #610181 and Cat #610404 were purchased from BD Transduction Laboratories (Lexington, KY). The Polyhema was purchased from Sigma (St. Louis, MO) while blasticin S and puromycin chemicals were obtained from Invitrogen (Carlsbad, CA). The full-length E-cadherin and Myc-tagged ZEB1 plasmid constructs and their corresponding empty vectors were purchased from Origene (Rockville, MD, USA).

RNA was extracted with TRIzol Reagent (Invitrogen) following manufacturer's protocol. The expression of genes was measured using real-time RT-PCR analyses with Taqman one-step RT-PCR reagents (Thermo Fisher Scientific) and results were normalized to co-amplified GAPDH. The primer and probe set for the following genes: FKBP5 (Hs01561006_m1), PIK3R1 (Hs00933163_m1), PIK3R2 (Hs00178181_m1), and GAPDH (4310884E) were purchased as inventoried mix (Applied Biosystems at Thermo Fisher). For immunoblotting, anti-AKT (Cell Signaling), anti-phosohoylated-473-AKT(Cell Signaling), anti-p85α (R&D), anti-p85β (R&D), anti-H3K4me2 (Milipore), anti-LSD1 (Abcam), anti-V5 (Sigma), anti-HDAC1 (Abcam), anti-GAPDH (Abcam), or anti-β-Tubulin (Abcam) antibodies were used. The inhibitors used are GSK2879552 (Selleck), ORY-1001 (Selleck), S2101 (Calbiochem), tranylcypromine (Calbiochem), and BKM120 (Selleck). Immunoblotting results shown are representative of at least 3 independent experiments.

The chromatin immunoprecipitation (ChIPs) assay kit (Cell Signaling Technology, Danvers, MA, USA) was used for ChIP assay according to protocol provided by the manufacturer’s instructions. In brief, the indicated cells were cultured on 100-mm culture dish around 70%~80% confluence and were fixed to cross-link proteins with DNA using 1% formaldehyde. The cell lysates were sonicated to shear DNA into small uniform fragments. Equal amounts of supernatants using anti-NKX2-8 (Abcam), or anti-HDAC1 (Abcam), or anti-H3K27ac (Abcam) and anti-IgG antibodies (Millipore, Billerica, MA) with protein G magnetic beads were immunoprecipitated overnight at 4°C. The cross-linked protein/DNA complexes were collected by magnetic pull down, and then were eluted from beads by elution buffer. PCR analysis with the indicated primers was conducted using the free DNA reversed from cross-linked protein/DNA complexes. The indicated ChIP primers are showed in Supplementary Table 1.