Protein samples were prepared from freshly dissected brain and used for semi-quantitative western blot analysis as previously described [33 (link)]. Primary antibodies were used in following conditions: anti-histone H3 (1:2,000; Abcam, Cambridge, MA), anti-acetyl histone H3 (1:2,000; Abcam, Cambridge, MA), anti-β-actin (1:10,000; Abcam, Cambridge, MA), anti-HDAC1 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-HDAC2 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-HDAC3 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-iba1 (1:2,000; Wako, Osaka, Japan). Properly matched secondary antibodies were used before ECL reaction: anti-mouse IgG (1:2,000; Vector Laboratories, Burlingame, CA), anti-rabbit IgG (1:10,000; Vector Laboratories, Burlingame, CA).
Anti hdac2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-HDAC2 is a laboratory reagent that specifically binds to and detects the HDAC2 protein. HDAC2 is a histone deacetylase enzyme involved in the regulation of gene expression. This product can be used to identify and quantify HDAC2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti hdac1, by Cell Signaling Technology (13 mentions)
Anti hdac3, by Cell Signaling Technology (6 mentions)
Anti gapdh, by Merck Group (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Anti h3k27ac, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti hdac6, by Cell Signaling Technology (3 mentions)
Anti histone h3, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti h3k27me3, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti h3k9ac, by Cell Signaling Technology (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Trametinib, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Rpmi culture medium, by Corning (2 mentions)
Anti yap1 antibody, by Abnova (2 mentions)
Anti hdac6, by Santa Cruz Biotechnology (2 mentions)
24 protocols using anti hdac2
1
Western Blot Analysis of Histone Modifications and HDAC Proteins
2
Chromatin Immunoprecipitation Antibody Panel
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anti-H3K27ac (Cat# 8173), anti-Batf (Cat# 8638), anti-H3K27me3 (Cat# 9733), anti-Mbd3 (Cat# 99169), anti-Chd3 (Cat# 4241), anti-Rbap46 (Cat# 6882), anti-Hdac1 (Cat# 34589), anti-Hdac2 (Cat# 57156), anti-EEA1 (Cat# 3288), anti-H3 (Cat# 4499), and anti-β-actin (Cat# 3700) were from Cell Signaling Technology (Danvers, USA). Anti-Kcnt2 (Cat# bs-12177R) was from Bioss Antibodies (Beijing, China). Anti-CD127 (A7R34), anti-c-Kit (2B8), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD19 (1D3), anti-NK1.1 (PK136), anti-CD150 (mShad150), anti-CD34 (RAM34), anti-CD45 (30-F11), anti-CD90 (HIS51), anti-Sca-1 (D7), anti-CD25 (PC61.5), anti-Flt3 (A2F10), anti-α4β7 (DATK32), anti-RORγt (AFKJS-9), anti-NKp46 (29A1.4), anti-Gata3 (TWAJ), anti-KLRG1 (2F1), anti-PLZF (Mags.21F7), Lineage cocktail (88-7772-72), anti-CD48 (HM48-1), Anti-IL-17 (eBio17B7), and anti-CD16/32 (93) were purchased from eBiosciences (San Diego, USA). Anti-BrdU (600-401-C29) was purchased from ThermoFisher. Paraformaldehyde (PFA) and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. The IL-17 ELISA kit was purchased from eBiosciences.
3
Protein Expression Analysis in Cells
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Cells were homogenized in RIPA lysis buffer for protein extraction, supplemented with protease inhibitors (Thermo Scientific, USA). Denatured proteins were separated in 12% SDS–PAGE and then transferred onto nitrocellulose papers (Pall, USA). After blotting, nitrocellulose papers were incubated with specific antibodies. Primary antibodies: anti-ERα antibody D8H8, 1:2000 (Cell Signaling Technology, USA, #8644); anti-ERα F-10, 1:1000 (Santa Cruz Biotechnology, USA, #sc-8002), anti-ERα 1D5, 1:1000 (Invitrogen, USA, #MA5-13191), anti-ERα-36, 1:200 (Alpha Diagnostic International, USA, #ERA361-A); anti-β-actin 13E5, 1:2000 (Cell Signaling Technology, USA, #8457); anti-HDAC2, 1:1000 (Cell Signaling Technology, USA, #2540); anti-VDAC1 N-18 (Santa Cruz Biotechnology, USA, #sc-8828). Secondary antibodies (HRP conjugated): anti-rabbit, 1:5000 (Cell Signaling Technology, USA, #7074); anti-mouse, 1:2000 (Cytiva, USA, #RPN4201). Immunolabelling was visualized using ECL procedure (PerkinElmer, USA). Uncropped and unprocessed scans of the most important blots are supplied in the Source Data file. All blots derive from the same experiment and they were processed in parallel.
4
Protein Extraction and Western Blot Analysis
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Whole cell protein was extracted using home-made radioimmunoprecipitation assay buffer (50mM Tris-HCl(pH7.4), 150mM Sodium chloride, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS). Protein concentration was measured using Bio-Rad Protein Assay Dye Reagent Concentrate (#500-0006, Bio-Rad, Hercules, CA) and proteins were preserved at −20°C. 50–100 μg protein samples were injected into wells of SDS–PAGE gels, separated using electrophoresis and transferred to membranes. Membrane-bound proteins were identified using rabbit antibodies for anti-collagen I (#72026, Cell signaling, Danvers, MA), anti-GR-α (#12041, Cell signaling, Danvers, MA), anti-GR-β (ab3581, Abcam, Cambridge, UK), anti-HDAC2 (#57156, Cell signaling, Danvers, MA), followed by anti-rabbit–horseradish peroxidase antibody. β-Tubulin (#15115, Cell signaling, Danvers, MA) or Actin (#Mab1501, Millipore, Burlington, MA) were used as controls.
5
Characterization of DAB2IP and NLGN3 in Cells
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The DAB2IP expression plasmid was prepared as described previously [22 (link)], and stable clones were selected by G418 at 1000 µg/mL for 3–4 wk. Knock-down of DAB2IP gene was performed using pGIPZ-DAB2IP-lentiviral-shRNAmir and pGIPZ-non-silencing-lentiviral-shRNAmir purchased from Open Biosystems, according to the manufacturer’s protocol. Following infection, cells were selected by puromycin at 0.2 µg/mL for 3–4 wk. The NLGN3 expression plasmid (CAG-HA-NLGN3 WT) was acquired from Peter Scheiffele (Addgene plasmid #59318; http://n2t.net.addgene:59318 ; RRID:Addgene_59318) [35 (link)]. Primary antibodies used were as follows: anti-NLGN3 (NBP1–90080, N-terminal epitope) was purchased from NOVUS biologicals; anti-DAB2IP (ab87811), anti-NLGN3 (ab186307, C-terminal epitope), and anti-NRXN3 (ab230635) were from Abcam; and anti-EGFR (Cat#4267), anti-HDAC2 (Cat#57156), anti-HSP90 (Cat#4874), anti-phospho-β-Catenin (Cat#9565), anti-phospho-GSK-3β (Cat#5558), and anti-actin (Cat#4970) were purchased from Cell Signaling Technology. PE-conjugated mouse anti-human CD133 (566593) was purchased from BD Pharmingen.
6
Antibody Sources and Applications
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Antibodies used in this study and their sources are as follows: anti-TRPS1 (R&D Systems#AF4838), anti-HDAC2 (Cell Signaling Technology#5113), anti-H4K16ac (Millipore#39929), anti-H4 (Millipore#04–858), anti-USP4 (Cell Signaling Technology#2651), anti-actin (proteintech#60008–1-Ig), anti-HA (Biotool#B23402), anti-Flag (Sigma#F7425), anti-Myc (Biotool#B23402), anti-His (Cell Signaling Technology#12698), and anti-Gal4 (Santa Cruz#510).
7
Western Blot Analysis of p53 Signaling Pathway
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Cells were lysed in 1× SDS sample buffer supplemented with the protease inhibitor mixture (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein (30 μg) were separated on SDS/polyacrylamide gels and then transferred onto membrane filters (Merck Millipore, Amsterdam, the Netherlands). After blocking with 5% non‐fat dry milk, the membranes were probed with anti‐p53 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐phospho‐p53 at Ser‐15 (Cell Signaling Technology, Danvers, CA, USA), anti‐acetyl‐p53 at Lys‐373/382 (Upstate, Lake Placid, NY, USA), anti‐p21WAF1 (Santa Cruz Biotechnology), anti‐Bcl‐2‐associated X protein (BAX; Cell Signaling Technology), anti‐NOXA (Cell Signaling Technology), anti‐HDAC2 (Cell Signaling Technology), anti‐poly (ADP‐ribose) polymerase (PARP; Cell Signaling Technologies), anti‐γH2AX (BioLegend, San Diego, CA, USA), anti‐ATM (Santa Cruz Biotechnology), anti‐phospho‐ATM at Ser‐1981 (Merck Millipore) or with anti‐actin antibody (Santa Cruz Biotechnology) followed by an incubation with horseradish peroxidase‐conjugated secondary antibodies (Invitrogen). Immunodetection was performed with enhanced chemiluminescence (ECL; GE Healthcare Life Science, Piscataway, NJ, USA).
8
Mycobacterium smegmatis Infection of Macrophages
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Mycobacterium smegmatis mc2155 was grown in Middlebrook’s 7H9 broth medium (Difco, New Jersey, USA) containing 0.05% Tween 80, 0.5% glucose and 0.5% albumin at 37°C on a shaker at 120 rpm. Murine RAW264.7 macrophage cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM; HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% L-glutamine. The cells were seeded onto 24-well and 6-well culture dishes at a density of 2x105 cells/ml and 1x107 cells/ml, respectively and proceeded for experiments. Anti- ATG5, anti-ATG7, anti-Beclin1, anti- H3K9me3, anti- H3K27me3, anti- H3K9ac, anti- H3K27ac, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-phospho–p38, anti-p62/SQSTM1, anti-GAPDH, anti-β-actin, and secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Cell Signaling Technologies (Massachusetts, USA). Anti-LC3I/II antibody was purchased from Sigma (Missouri, USA). All the pharmacological inhibitors were purchased from Sigma (Missouri, USA) and Calbiochem (Massachusetts, USA) and reconstituted in DMSO (Himedia, Mumbai, India) or sterile H2O at the following concentrations: U0126 (10 µM), SB203580 (10 µM), UNC0638 hydrate (5µM), rapamycin (50nM) and 3MA (10mM).
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Mycobacterium smegmatis mc2155 was grown in Middlebrook’s 7H9 broth medium (Difco, New Jersey, USA) containing 0.05% Tween 80, 0.5% glucose and 0.5% albumin at 37°C on a shaker at 120 rpm. Murine RAW264.7 macrophage cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM; HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% L-glutamine. The cells were seeded onto 24-well and 6-well culture dishes at a density of 2x105 cells/ml and 1x107 cells/ml, respectively and proceeded for experiments. Anti- ATG5, anti-ATG7, anti-Beclin1, anti- H3K9me3, anti- H3K27me3, anti- H3K9ac, anti- H3K27ac, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-phospho–p38, anti-p62/SQSTM1, anti-GAPDH, anti-β-actin, and secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Cell Signaling Technologies (Massachusetts, USA). Anti-LC3I/II antibody was purchased from Sigma (Missouri, USA). All the pharmacological inhibitors were purchased from Sigma (Missouri, USA) and Calbiochem (Massachusetts, USA) and reconstituted in DMSO (Himedia, Mumbai, India) or sterile H2O at the following concentrations: U0126 (10 µM), SB203580 (10 µM), UNC0638 hydrate (5µM), rapamycin (50nM) and 3MA (10mM).
9
Western Blot Analysis of Histone Deacetylases
10
Regulation of IRF7 Signaling Pathway
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