For transactivation studies of HIF-1α, HIF-2α, and p300, the binary Gal4 reporter plasmids (HIF-1α-N-TAD, aa 530-778; HIF-1α-C-TAD, aa 740-826; HIF-2α-TAD, aa 819-870; p300-N-TAD, aa 1-596; p300-C-TAD, aa 1737-2414) were provided by Dr. Nianli Sang, Drexel University College of Medicine. pFR-Luc (Stratagene) reporter contains the yeast Gal4-binding site upstream of a minimal promoter and the Firefly luciferase gene. Enolase1-WT and Enolase1-HRE-mut promoter were provided by Dr. Gregg Semenza, Johns Hopkins University. HDAC1 expression construct was provided by Dr. Stuart Schreiber, Harvard University (22 (link)). HDAC2 and HDAC3 were provided by Dr. Ed Seto, H. Lee Moffitt Cancer Center Research Institute (23 (link), 24 (link)). HRE-Luc (#26731) by Navdeep Chandel; HDAC4 (#30485), HDAC6 (#30482) and HDAC6-DC (#30483) by Tso-Pang Yao, and ODD-luciferase-pcDNA3 by William Kaelin (#18956) were obtained from Addgene. pRLTK (Promega) containing the Renilla reniformis luciferase gene was used as an internal transfection control. PHD2f/f;CreER(+) and PHD2+/+;CreER(+) MEFs were a kind gift from Dr. William G. Kaelin of Harvard Medical School (25 (link)).
Hdac4
Manufactured by Addgene
Sourced in United States
HDAC4 is a histone deacetylase enzyme that plays a role in the regulation of gene expression. It removes acetyl groups from lysine residues on histone proteins, leading to a more compact chromatin structure and reduced gene transcription. HDAC4 is involved in various cellular processes, including cell differentiation, development, and metabolism.
Automatically generated - may contain errors
3 protocols using hdac4
1
Transactivation Assays for HIF and p300
2
Overexpression of HDAC4 and DDIT4 in Human Fibroblasts
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The mammalian expression plasmids for HDAC4 (plasmid #30485) and DDIT4 (RC202847) were purchased from Addgene (Cambridge, MA, USA) and OriGene (Rockville, MD, USA), respectively. Each plasmid was transiently transfected into human dermal fibroblasts using jetPRIME® by following the manufacturer's instructions. HDAC4 lentiviral particles were produced according to the manufacturer’s protocol (System Biosciences, Mountain View, CA, USA) using pCDH-CMV-MCS-EF1-Puro plasmid or pCDH-CMV-HDAC4-EF1-Puro plasmid. The pCDH-CMV-HDAC4-EF1-Puro plasmid was produced by amplifying the HDAC4 cDNA from pcDNA3.1-HDAC4 plasmid by PCR and subcloning it into pCDH-CMV-MCS-EF1-Puro plasmid. Briefly, HEK-293TN cells were transfected with plasmids containing pGag-pol and pVSV-G using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The viral supernatant was collected and transduced into the human dermal fibroblasts. Infected cells were selected using 5 μg/mL puromycin (SNUH-IBC-1903-007-004).
3
Plasmid Engineering and Transfection
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Plasmids encoded with VHL, 14-3-3ζ, HDAC4, HDAC5, FoxO1, CFL1, BICRA were obtained from Addgene. Difopein DNA was constructed with 3 ssDNA oligos and 3 primers (sequence can be found in Table S1) by overlapping PCR. Plasmids encoded with VHL 3 KR-14-3-3ζ (1–230) 20 KR mutation, VHL 3 KR-14-3-3ζ (1–230) 19 KR K49E mutation were synthesized by GenScript Biotech. The transfection vectors of each gene described above were constructed into pcDNA3.1 backbone using PCR amplification.
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