Anti acetyl histone h3 lys9

Anti-p300 (dilution 1:100, N-15; sc-584), anti-CBP (dilution 1:100, A-22; sc-369), anti-PCAF (dilution 1:100, E-8; sc-13124), anti-cyclin B1 (dilution 1:100, GNS1; sc-245), and anti-CDK1 (dilution 1:200, 17; sc-54) antibodies were obtained from Santa Cruz Biochemistry (Santa Cruz, CA). Anti-caspase 3 (dilution 1:1000, 8G10; #9662), anti-cleaved caspase 3 (dilution 1:500, 5A1E; #9664), anti-cleaved PARP (dilution 1:1000, D64E10; #5625), anti-phospho-histone H3 (Ser10) (dilution 1:1000, D2C8; #3377), anti-acetyl histone H3 (Lys9) (dilution 1:1000, C5B11; #9649), anti-acetyl histone H3 (Lys18) (dilution 1:1000, D8Z5H; #13998), anti-acetyl histone H3 (Lys27) (dilution 1:1000, D5E4; #8173), and anti-histone H3 (dilution 1:2000, D1H2; #4499) antibodies were from Cell Signaling Technology (Danvers, MA). Anti-β-actin antibody (dilution 1:2000, AC-15; A5441) was from Sigma-Aldrich (St. Louis, MO). The HAT inhibitor C646 was obtained from Sigma-Aldrich (SML0002) and Selleck Chemicals (S7152) (Houston, TX).

Cells were lysed in boiling Laemmli buffer supplemented with protease inhibitors, then sonicated and complemented with DTT. Protein concentration was determined by BCA (Thermo Scientific) assay. Fifteen to twenty micrograms of total protein were loaded onto SDS-PAGE gels and transferred onto nitrocellulose membranes. The membrane was blocked with TBST (1× TBS with 0.1% Tween 20) + 5% milk at room temperature for 1 h and incubated with primary antibody and then with horseradish peroxidase (HRP)-coupled secondary antibody (Amersham, Piscataway, NJ, United States). Activity was visualized by electrochemiluminescence. Antibodies used in this study are anti-Histone H3 (Cell signaling Technology #9717) and anti-Acetyl-Histone H3 (Lys9) (Cell signaling Technology #9649).

Two kinds of class I and II HDACis, TSA and SAHA, were obtained from Union Biomed (Taipei, Taiwan); recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA); the anti-ANKH and anti-α-tubulin antibodies were obtained from Novus Biologicals (Littleton, CO, USA); the class I HDAC antibody sampler kit, class II HDAC antibody sampler kit, anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-histone H3, anti-acetyl-histone H3-Lys9, and anti-acetylated-lysine (Ac-K-103) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA); the anti-ENPP1, anti-TNAP, anti-acetyl-histone H4, anti-TGF-β receptor I (TGFBR1), and anti-TGF-β receptor II (TGFBR2) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP), and anti-rabbit IgG HRP antibodies were obtained from GeneTex International (Hsinchu City, Taiwan).

Total cellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH 8.0 containing 1% NP-40, 150 mM NaCl, 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride) containing protease and phosphatase inhibitors. The total protein was quantified using Bradford reagent. Equal amounts of protein lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with 5% skimmed milk for 2 h at room temperature. The blots were incubated with different primary antibodies overnight at 4 °C, followed by the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000) for protein visualization with ECL reagents (Millipore). We utilized the following primary antibodies: anti-ACLY (Cell Signaling, 4332), anti-ACeCS1 (Cell Signaling, 3658), anti-SAT1 (Cell Signaling, 61586), anti-SP1 (Cell Signaling, 9389), anti-acetyl-lysine (Cell Signaling, 9441), anti-β-actin (DHSB, JLA20), anti-acetyl-histone H3 (Lys9) (Cell Signaling, 9649), anti-acetyl-histone H3 (Lys27) (Cell Signaling, 4353), anti-acetyl-histone H3 (Lys18) (Cell Signaling, 9675), anti-histone H3 (Abcam, ab1791), anti-ACLY (Abcam, ab40793), anti-H3K27 (Active Motif, 39133) and HA antibody (Covance, MMS-101R). Protein expression levels were normalized to the respective loading control, β-actin.

Mice trained in vivo by injection of β‐glucan (25 mg kg⁻¹) and untrained mice, as above, were sacrificed after 7 d. Their lungs were isolated and treated with collagenase and dispase to yield single‐cell suspensions. After lysing RBCs, the cells were fixed with by incubation with CellCover solution (Anacyte Laboratories, 800‐125) for 10 min at room temperature and centrifuged at 500 x g for 5 min. After discarding the supernatant, cell fixation and permeabilization solution (Invitrogen, 005523‐00) was added to each pellet, followed by vortexing and incubation for 1 h at room temperature or 4 °C. The cells were washed, and staining buffer (eBioscience Foxp3/Transcription staining buffer set) was added, followed by the addition of antibodies (1:50) and incubation for 30 min at room temperature. These antibodies included anti‐histone H3 (D1H2, 12167S), anti‐tri‐methyl‐histone H3 (K4) (C42E8, 12064S), anti‐acetyl‐histone H3 (Lys 9) (C5B11, 4484S), and anti‐acetyl‐histone H3 (Lys27) (D5E4, 15485S), all from Cell Signaling. Cells were classified as epithelial cells by their expression of EpCAM and as macrophages by their expression of Siglec F. After washing, these cells were subjected to flow cytometry.