Hdac6

Manufactured by Abcam

Sourced in United Kingdom

HDAC6 is a histone deacetylase enzyme that catalyzes the removal of acetyl groups from lysine residues on histone proteins. This enzyme plays a role in regulating gene expression and cellular processes.

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HDAC6 activity assay kit HDAC6 antibody Fluorometric HDAC6 activity assay kit HDAC6 activity kit lysis buffer Anti-HDAC6

7 protocols using hdac6

Antibody Generation and Validation for Acetylation-Specific HMGN2

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Rabbit polyclonal ac-K2-HMGN2 antibody was made by New England Peptides. The antibody was raised against an acetylated N-terminal HMGN2 peptide (P(KAc)RKAEGDAC), followed by affinity purification. To ensure specificity, a blocking peptide (P(KAc)RKAEGDAC) and a non-acetyl blocking peptide (PKRKAEGDAC) were used to ensure acetyl-specific interaction. Antibodies used for western blotting; Tubulin (Invitrogen, 322500, 1:2000); HDAC6 (Abcam, ab47181, 1:500); HMGN2 (Millipore, 07-252, 1:500; Cell Signaling, #9437, 1:2000) pan-acetyl lysine (Millipore, AB3879, 1:500); CISH (Santa Cruz, sc-15344, 1:500); CyclinD1 (Santa Cruz, sc-718, 1:500); CEBPβ (Santa Cruz, sc-150, 1:500); PARP/Cleaved PARP (Cell Signaling, #9542, 1:500); ac-K2-HMGN2 (1:50); acetyl-α-Tubulin (Cell Signaling, #5335, 1:500). Secondary antibody was used at a dilution of 1:1000 for all antibodies except Tubulin, which was 1:2000. Antibodies used for IHC: CyclinD1 (Santa Cruz, sc-718, 1:1500); acetyl-Tubulin (Cell Signaling, D20G3, 1:100); ac-K2-HMGN2 (1:500).

Medler T.R., Craig J.M., Fiorillo A.A., Feeney Y.B., Harrell J.C, & Clevenger C.V. (2016). HDAC6 Deacetylates HMGN2 to Regulate Stat5a Activity and Breast Cancer Growth. Molecular cancer research : MCR, 14(10), 994-1008.

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Western Blot Analysis of HDAC6

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Cells were solubilized in ice-cold RIPA buffer (Invitrogen) supplemented with complete protease inhibitor tablets (Roche, Basel, Switzerland). 40 μg of total protein was resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blotted with antibodies against HDAC6 (Abcam, Cambridge, UK) and α-tubulin (Cell Signaling Technology, Danvers, MA, USA) and HRP-conjugated secondary antibodies. The protein bands were visualized by the ECL system according to the manufacturer’s instructions and exposed to x-ray film.

Wang Y., Shi Z.Y., Feng J, & Cao J.K. (2018). HDAC6 regulates dental mesenchymal stem cells and osteoclast differentiation. BMC Oral Health, 18, 190.

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Quantifying mitotic phosphoproteome

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GFP (Clontech, Mountain View, CA, USA, 632677), H3 (Abcam plc., Cambridge, UK, ab1791), Haspin (Abcam plc. ab226222), Vinculin (Sigma-Aldrich V9131), HDAC6 (Abcam plc. ab1440), Dido3 (Merck-Millipore, Burlington, MA, USA, ABC480), acetylated tubulin (Sigma-Aldrich T7451), gamma-tubulin (Sigma-Aldrich T5192), Ki-67 (Thermo-Fisher Scientific SolA15), H3 Thr3 phosphorylated (Upstate 05-746), AlexaFluor goat anti-mouse 488 (Thermo-Fisher Scientific A28175), AlexaFluor goat anti-rabbit 488 (Thermo-Fisher Scientific A11034), AlexaFluor goat anti-mouse 594 (Thermo-Fisher Scientific A11032), AlexaFluor Goat anti-mouse 647 (Thermo-Fisher Scientific A21235).

Quadri R., Sertic S., Ghilardi A., Rondelli D., Gallo G.R., Del Giacco L, & Muzi-Falconi M. (2021). Phosphorylation of H3-Thr3 by Haspin Is Required for Primary Cilia Regulation. International Journal of Molecular Sciences, 22(14), 7753.

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Construction and Use of GFP-HDAC6 Expression Plasmid

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The GFP-HDAC6 expression plasmid was constructed by cloning human HDAC6 cDNA into the pEGFPN1 vector, and the catalytically inactive mutant was generated by site-directed mutagenesis as described (Zhou et al., 2009 (link); Li et al., 2014 (link)). Triethylammonium bicarbonate, formaldehyde-H₂ (28% in H₂O), sodium cyanoborohydride, ammonium hydroxide, trifluoroacetic acid, 4’,6-diamidino-2-phenylindole (DAPI), fluorescein-phalloidin, trichostatin A (TSA), and sodium butyrate (NaB) were purchased from Sigma-Aldrich. Formaldehyde-D₂ (28% in D₂O, Cambridge Isotope Laboratory), formic acid and acetonitrile (Merck), and C₁₈ desalting microcolumns (Nest Group) were from the indicated sources. Tubacin was obtained from Stuart Schreiber (Harvard Medical School). Antibodies against α-tubulin, acetylated α-tubulin, and β-actin (Sigma-Aldrich), HDAC6, HDAC7, HDAC9, Hsp90, Prx I, MYH9, Hsc70, and DNAJA1 (Abcam), HDAC3, HDAC4, HDAC5, HDAC11, and SIRT2 (Santa Cruz Biotechnology), acetylated lysine (Cell Signaling Technology), and GFP (Roche), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), and rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were obtained from the indicated sources.

Zhang L., Liu S., Liu N., Zhang Y., Liu M., Li D., Seto E., Yao T.P., Shui W, & Zhou J. (2014). Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity. Protein & Cell, 6(1), 42-54.

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Immunofluorescence Staining of Liver Sections and Cell Lines

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Confocal IF microscopy was performed with a Zeiss LSM 510 confocal microscope using the 63X oil objective as previously described (see Supporting Methods). Briefly, unstained liver sections were deparaffinized and rehydrated, boiled in antigen unmasking solution, and quenched with Image-IT FX signal enhancer (Invitrogen, Grand Island, NY). Normal human cholangiocytes and CCA cells lines were washed with PBS and fixed in ice cold methanol for 5 min at −20°C. Slides were blocked for 1 hour at room temperature and then incubated overnight at 4°C with acetylated α-tubulin (1:500, Sigma-Aldrich), γ-tubulin (1:500, Sigma-Aldrich), and/or HDAC6 (1:100, Abcam) antibodies followed by 1 hour incubation with fluorescent secondary antibodies (1:100). Nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI; Prolong Gold w/DAPI, Invitrogen).

Mansini A.P., Lorenzo Pisarello M.J., Thelen K.M., Cruz-Reyes M., Peixoto E., Jin S., Howard B.N., Trussoni C.E., Gajdos G.B., LaRusso N.F., Perugorria M.J., Banales J.M, & Gradilone S.A. (2018). MiR-433 and miR-22 dysregulations induce HDAC6 overexpression and ciliary loss in cholangiocarcinoma. Hepatology (Baltimore, Md.), 68(2), 561-573.

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Quantification of Cytoskeletal Regulators

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Whole-cell lysates were collected in the Laemelli sample buffer as previously described [18 (link)]; nuclear and cytoplasmic fractions were isolated as previously described [19 (link)]. Equal volumes of lysate were blotted and quantified as previously described [2 (link),18 (link),19 (link),20 (link)]. Three pairs of lysates were used to determine the levels of detyrosinated α-tubulin (Abcam, Cambridge, UK), HDAC6, ATAT1 (Abcam), SIRT2 (CST), TGM2 (CST), and HSP90α (CST) expression levels. Whole-cell lysates were collected from BoM-MDA-231 cells following growth under standard culture conditions and blotted. For all western blots conducted using cytoplasmic and whole-cell lysate, β-actin (CST) was used as a loading control. Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA) was utilized as a loading control for western blots involving nuclear protein fractions.

Othman A., Winogradzki M., Patel S., Holmes W., Blank A, & Pratap J. (2022). The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells. Cancers, 14(14), 3436.

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Protein Expression Analysis by Western Blot

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Cells were harvested and lysed in lysis buffer containing protease inhibitors (Roche). Total protein (10–30 μg) was immunoblotted with specific primary antibodies; HDAC6 (Abcam), Pax7 (DSHB), HIF1α (34 (link)), HIF2α (Novus), Acetyl-H3, Acetyl-H4 (Millipore). α-Tubulin (Calbiochem) was used as an internal control. Quantification of band intensity was analyzed using TINA 2.0 (RayTest) or ImageJ (NIH) and normalized to the intensity of α-tubulin.

Lee S.W., Yang J., Kim S.Y., Jeong H.K., Lee J., Kim W.J., Lee E.J, & Kim H.S. (2015). MicroRNA-26a induced by hypoxia targets HDAC6 in myogenic differentiation of embryonic stem cells. Nucleic Acids Research, 43(4), 2057-2073.

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