Recombinant Human HDAC5 protein, GST-tagged

• Cat.No.: HDAC5-28267TH
• Product Overview: Recombinant Human full length HDAC5, fused with GST tag at N-terminal, was expressed in Insect cells.

Specification

• Species: Human
• Source: Insect Cells
• Tag: GST
• Form: 40 mM Tris-HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, 16 mM glutathione, 20% glycerol
• Bio-activity: ≥2500 pmol/min/µg.
• Molecular Mass: 150 kDa
• Purity: >8%
• Unit Definition: One U =1 pmol of acetyl group removed/min/µg of enzyme
• Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
• Storage: >6 months at -80 centigrade. Avoid freeze/thaw cycles.
• Concentration: 0.04 mg/m

Gene Information

• Gene Name: HDAC5 histone deacetylase 5 [ Homo sapiens ]
• Official Symbol: HDAC5
• Synonyms: HDAC5; histone deacetylase 5; FLJ90614; KIAA0600; NY CO 9; antigen NY-CO-9; HD5; NY-CO-9
• Gene ID: 10014
• mRNA Refseq: NM_005474
• Protein Refseq: NP_005465
• MIM: 605315
• UniProt ID: Q9UQL6
• Chromosome Location: 17q21
• Pathway: B Cell Receptor Signaling Pathway, organism-specific biosystem; Cell cycle, organism-specific biosystem; MicroRNAs in cardiomyocyte hypertrophy, organism-specific biosystem; NOTCH1 Intracellular Domain Regulates Transcription, organism-specific biosystem; Signal Transduction, organism-specific biosystem; Signaling by NOTCH, organism-specific biosystem; Signaling by NOTCH1, organism-specific biosystem
• Function: NAD-dependent histone deacetylase activity (H3-K14 specific); NAD-dependent histone deacetylase activity (H3-K9 specific); NAD-dependent histone deacetylase activity (H4-K16 specific); histone deacetylase activity; histone deacetylase activity (H3-K16 specific); hydrolase activity; protein binding; protein kinase C binding; repressing transcription factor binding; transcription factor binding; contributes_to transcription regulatory region DNA binding

Citation

Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

Journal: Oncogene    PubMed ID: 27212032    Data: 2016/4/17

Authors: Chunyu Cao, Shauna N. Vasilatos, Yi Huang

Article Snippet:Pull-down of IgG was used as negative control.Pull-down of IgG was used as negative control.. 0.25 μg of recombinant human GST-tagged HDAC5 protein (Creative BioMart, NY, NY) was mixed with 30 μl immunoprecipitates or 1.5 μg bulk histone at 37°C for 6h in a buffer containing 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 50 mM NaCl, 2 mM KCl, 0.5mM DTT, 1mM EDTA and protease inhibitor.. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody (EMD Millipore, Billerica, MA).The reactions were then subjected to immunoblots with anti-acetyl lysine antibody (EMD Millipore, Billerica, MA).

Correlated overexpression of HDAC5 and LSD1 protein in breast cancer. (a) The levels of mRNA expression of HDAC5 and LSD1 in breast cancer cell lines versus MCF10A cells (set as fold 1) using real-time qPCR with β-actin as an internal control. (b) Immunoblots with anti-HDAC5 and LSD1 antibodies in indicated cell lines. β-actin protein was blotted as a loading control. (c) Histograms represent the mean protein levels of HDAC5 or LSD1 in three determinations relative to β-actin ± s.d. as determined by quantitative immunoblots. (d) 50 primary human invasive breast tumor samples were immunostained with antibodies against HDAC5 or LSD1. Chi-square study was performed by using median H-scores as the cutoff for high vs low protein expression. (e) Representative HDAC5 and LSD1 staining (200x) in invasive breast carcinoma and adjacent normal tissue specimens from one representative patient. H-scores represent average staining intensity in breast tumors (n=18) versus adjacent normal breast tissue (n=18). (f) Representative HDAC5 and LSD1 staining (200x) in stage 2 and 3 invasive breast carcinoma specimens. H-scores represent average staining intensity in stage 3 breast tumors (n=25) versus stage 2 breast tumors (n=25). * p <0.05, ** p <0.01, *** p <0.001, Student’s t-test.

HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.

HDAC5 stabilizes LSD1 protein in breast cancer cells. (a) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 48 h. mRNA expression of HDAC5 and LSD1 was measured by quantitative real-time PCR with β-actin as an internal control. (b) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h. Effect of HDAC5 overexpression on LSD1 protein expression in MDA-MB-231 cells was evaluated by immunoblots with anti-LSD1 and anti-HDAC5 antibodies. (c) MDA-MB-231 cells were transfected with scramble siRNA or HDAC5 siRNA for 48 h. Effect of HDAC5 knockdown on LSD1 mRNA expression was examined by quantitative real-time PCR with β-actin as internal control. (d) Effect of HDAC5 siRNA on LSD1 protein expression in MDA-MB-231 cells. (e) Effect of depletion of LSD1 on mRNA expression of HDAC5 in MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells. (f) Effect of LSD1-KD on protein expression of HDAC5 in MDA-MB-231-scramble or MDA-MB-231-LSD1-KD cells. (g) MDA-MB-231 cells were transfected with control vector pcDNA3.1, pcDNA3.1-HDAC5, scramble siRNA or HDAC5 siRNA for 48 h and analyzed by immunoblots for nuclear expression of indicated histone marks. PCNA was used as loading control. (h) Effect of HDAC5 overexpression or siRNA on LSD1 protein half-life in cycloheximide chase study. (i) Measurement of LSD1 half-life using Calcusyn program. (j) Effect of siRNA knockdown of LSD1 cofactors or class II HDACs on LSD1 protein level. All the experiments were performed three times. Bars represent the mean of three independent experiments ± s.d. * p <0.05, ** p <0.01, *** p <0.001, Student’s t-test.