Hdac1

Manufactured by Thermo Fisher Scientific

Sourced in United States

HDAC1 is a laboratory instrument used for the detection and analysis of histone deacetylase 1, an enzyme involved in the regulation of gene expression. The core function of HDAC1 is to measure the activity and levels of this enzyme in biological samples.

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Lab products found in correlation

9 protocols using hdac1

Silencing Key Regulators in MM Cells

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Short interfering (si)RNA for BIM, BAX, BAK, HDAC1, HDAC2 and HDAC6 were purchased from Thermo Fisher Scientific. ERK1 and ERK2 siRNA were purchased from Santa Cruz Biotechnology. siRNA were electroporated into MM cell lines using the Lonza nucleofector kit V (Lonza, Basel, Switzerland). The manufacturer’s G-15 program was used for KMS18 and OPM2; O-23 was used for MM1S and KMS28. All experiments were performed in triplicate.

Ramakrishnan V.G., Miller K.C., Macon E.P., Kimlinger T.K., Haug J., Kumar S., Gonsalves W.I., Rajkumar S.V, & Kumar S.K. (2019). Histone deacetylase inhibition in combination with MEK or BCL-2 inhibition in multiple myeloma. Haematologica, 104(10), 2061-2074.

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Immunoblotting and ChIP Antibody Protocols

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The following antibodies were used for immunoblotting; p16 (clone JC8, sc-56330, Santa Cruz Biotechnology), p14 ARF (Santa Cruz sc-8613), beta Actin (Santa Cruz, sc-69879), Flag (Sigma, F1804), AICDA (Cell signaling, #4975), SMAD3 (Invitrogen, 51–1500), Rb1 (cell signaling, #9309), SETD7 (Cell signaling, #2825), and alpha Tubulin (Sigma, T6074), and chromatin immunoprecipitation; CtBP1 (EpiGentek, #A2705), HDAC1 (Thermo Fisher PA1-860), and p300 (Bethyl A300-358A) and p53 (Sant Cruz, sc126).

Ohashi M., Hayes M., McChesney K, & Johannsen E. (2021). Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes. PLoS Pathogens, 17(3), e1009419.

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Protein Expression Profiling in Cell Lysates

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Cell lysates were prepared using a buffer containing 10 mmol/L Tris-HCl at pH 7.4 and 1% sodium dodecyl sulfate (SDS). Protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed on the cell lysates, transferred to polyvinylidene fluoride membranes, and immunostained overnight using antibodies as described below. The following antibodies were purchased from Cell Signaling Technology: p21 (dilution at 1:1,000), p27 (1:1,000), cyclinB1 (1:1,000), aurora kinase A (AURKA; 1:1,000), polo-like kinase 1 (PLK1; 1:1,000), cyclin-dependent kinase 1 (CDK1; 1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), acetyl-histone H3 (1:1,000), histone H3 (1:1,000), p44/p42 MAPK (ERK1/2; 1:1,000), p-p44/p42 MAPK (ERK1/2; 1:1,000), p-AKT1 (1:1,000), and AKT1 (1:1,000). Aurora kinase B (AURKB; 1:1,000) and HDAC2 (1:1,000) antibodies were purchased from Abcam. Survivin (1:1,000) was obtained from Novus Biologicals, N-cadherin (1:1,000) from EMD Millipore, HDAC1 (1:1,000) from Thermo Fisher Scientific, and HDAC10 from BioVision. The following antibodies were purchased from Santa Cruz Biotechnology: p27(1:1,000), HDAC4(1:1,000), HDAC6 (1:1,000), and GAPDH (1:5,000). Band densitometry analysis was performed using ImageJ software (NCI).

Kotian S., Zhang L., Boufraqech M., Gaskins K., Gara S.K., Quezado M., Nilubol N, & Kebebew E. (2017). Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits Thyroid Cancer Growth and Metastases. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5044-5054.

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Epigenetic Regulators in Protein Interactions

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IP was performed33 (link) using nuclear fraction buffer and antibodies (ASXL2 at a dilution of 1:500, Santa Cruz, sc-169972; HDAC1 at a dilution of 1:500, ThermoFisher Scientific, PA1-860; and HDAC2 at a dilution of 1:2,000, PA1-861). After washing with IP buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mM NaF and protease inhibitor cocktail (Roche)) for four times, the associated proteins were collected for western blot analysis34 . The histone modifications and protein levels were determined using antibodies at dilutions of 1:800 against H3K4me3 (Abcam, ab8580) and H3K27me3 (ab6002).

Li J., He F., Zhang P., Chen S., Shi H., Sun Y., Guo Y., Yang H., Man N., Greenblatt S., Li Z., Guo Z., Zhou Y., Wang L., Morey L., Williams S., Chen X., Wang Q.T., Nimer S.D., Yu P., Wang Q.F., Xu M, & Yang F.C. (2017). Loss of Asxl2 leads to myeloid malignancies in mice. Nature Communications, 8, 15456.

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Antibody Immunoblotting Protocol

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The antibodies were obtained commercially from various sources – 1) Cell Signaling and Technology -Lamin A/C ( #4777); EpCAM (#2626S); DNMT1 (#5032P); HDAC1 (#5356P); G9a (3306S); Snail1 (3879P); α4-Integrin (#8440S); β-actin (#4970S); Di, Tri-methyl histone H3 (Lys9) (#5327) 2) Thermoscientific – UHRF1 (PA5-27969) 3) BD Biosciences – E-cadherin (610181); p120-catenin (610133); β-catenin (610154) 4) Bethyl laboratories – MARVELD2 (A301-505A) and 5) Sigma/Aldrich – β-tubulin (T0198).

Duvall-Noelle N., Karwandyar A., Richmond A, & Raman D. (2015). LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex. Oncogene, 35(9), 1122-1133.

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Protein Expression Analysis Protocol

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For analysis of soluble proteins, cells or tissues were homogenized in RIPA lysis buffer, and the total protein content was extracted as detailed in a previously described protocol (Wang et al, 2012). The extracts were analyzed by 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by blotting with the following antibodies: E2F1 (Abcam, ab137415, 1:1,000), Cyclin A (Santa Cruz, H‐432, 1:2,000), PCNA (Abcam, ab15497, 1:1,000), P21 (Abcam, ab7960, 1:2,000), TDP‐43 (Proteintech, 10782‐2‐AP; Rosemont, IL, 1:1,000), HDAC1 (Thermo, PA1‐860; Waltham, MA, 1:1,000), γH2AX (Millipore, 05‐636, 1:800), acetyl‐histone H3 (Santa Cruz, SC‐8655, 1:500), total histone H3 (Cell Signaling, #4499, 1:1,000), and tubulin (Abcam, ab4074, 1:5,000). After primary antibody binding, the blots were incubated at room temperature with the appropriate secondary antibodies and Western Lightning Plus‐ECL (PerkinElmer; Waltham, MA). All cell data are repeated at least 3 independent experiments. For quantitative analysis, relative intensities of the bands were normalized against those of internal controls and expressed as mean ± SEM.

Wu C., Jin L., Wang I., Wei W., Ho P., Liu Y, & Tsai K. (2020). HDAC1 dysregulation induces aberrant cell cycle and DNA damage in progress of TDP‐43 proteinopathies. EMBO Molecular Medicine, 12(6), e10622.

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Antibody Immunoblotting Protocol

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Duvall-Noelle N., Karwandyar A., Richmond A, & Raman D. (2015). LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex. Oncogene, 35(9), 1122-1133.

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Investigating Epigenetic Regulation in Cancer

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Cisplatin (CDDP), paclitaxel (Taxel), doxorubicin (Dox), Vinorelbine (NVB) and SAHA were obtained from Sigma (St. Louis, MO, USA). Belinostat was synthesized in Medicine Chemistry Laboratory at Shenyang Pharmaceutical University (purity >97%). The primary antibodies against HDAC1, HDAC3, HDAC6, Nanog, Oct4, Sox2, ERK, phosphor-ERK and β-actin were got from Cell Signaling Technology (Beverly, MA, USA). The primary antibodies against CD133 was obtained from Mitenyi Biotec. The primary antibodies against TRIB1, C/EBP-β, HDAC8 and Acetyl-p53 were purchased from Abcam (Cambridge, MA, USA). Antibodies to ALDH1A1, ALDH1A3, ALDH2, ALDH3A1, ALDH5A1 and ALDH7A1 were obtained from Novus Biologicals (Littleton, CO, USA). The Silencer Select Validated siRNA, including TRIB1, HDAC1 and C/EBP-β, were got from Life technologies (Gaithersburg, MD, USA).

Wang L., Liu X., Ren Y., Zhang J., Chen J., Zhou W., Guo W., Wang X., Chen H., Li M., Yuan X., Zhang X., Yang J, & Wu C. (2017). Cisplatin-enriching cancer stem cells confer multidrug resistance in non-small cell lung cancer via enhancing TRIB1/HDAC activity. Cell Death & Disease, 8(4), e2746-.

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Plasmid Transfection and Gene Silencing

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The pcDNA plasmid was a gift from Professor Chio Oka (NARA Institute of Science and Technology, ikoma, Japan), the pCMX-hRXR-α and pBJ5-HDAC plasmids were a gift from Professor Makoto Makishima (Nihon University, Tokyo, Japan), and the pGL3-basic and phRL-tk plasmids were obtained from Yingrun Biotechnologies (Changsha, China). HDAC1, RXRα, HtrA1 and control siRNA were obtained from Life Technologies (Waltham, MA, USA), RXRγ siRNA was obtained from RIBOBIO (Guangzhou, China). Cells were transfected with plasmids using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h and siRNA using Lipofectamine RNAi MAX (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h, followed by the next treatment.

Wang W., Zhao M., Cui L., Ren Y., Zhang J., Chen J., Jia L., Zhang J., Yang J., Chen G., Ashby CR J.r., Wu C., Chen Z.S, & Wang L. (2020). Characterization of a novel HDAC/RXR/HtrA1 signaling axis as a novel target to overcome cisplatin resistance in human non-small cell lung cancer. Molecular Cancer, 19, 134.

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