Pan acetyllysine

Manufactured by Cell Signaling Technology

Sourced in China

Pan-acetyllysine is a laboratory reagent used for the detection and enrichment of acetylated lysine residues in proteins. It provides a general tool for the study of protein acetylation, a post-translational modification that regulates various cellular processes.

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Lab products found in correlation

Flag tag (flag), by Merck Group (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Merck Group (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Proteinase inhibitor, by Roche (1 mentions) Halt protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Nicotinamide, by Merck Group (1 mentions) Smartpool sirna, by Horizon Discovery (1 mentions) Gsk690693, by Selleck Chemicals (1 mentions) Azd5363, by Selleck Chemicals (1 mentions) Palbociclib, by Selleck Chemicals (1 mentions) Mk 2206, by Selleck Chemicals (1 mentions) Sirt3, by Cell Signaling Technology (1 mentions) Pycr1, by Proteintech (1 mentions) Flag peptide, by Merck Group (1 mentions) α tubulin, by Santa Cruz Biotechnology (1 mentions)

8 protocols using pan acetyllysine

Quantification of Histone Modifications

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Pulverized tumor tissue was weighed (10mg) and proteins were extracted with RIPA buffer (Millipore:20–188) containing proteinase inhibitor (Roche: 4693159001) for 1 hour, followed by centrifuging at 16,000g for 10 mins to get supernatant. Protein concentrations were determined using Pierce BCA protein assay kit (ThermoFisher#23225). Around 20 μg of protein was loaded (mixed with loading buffer) into 4–20% gel (Bio-Rad: 4568095), transferred onto membrane, incubated with primary antibodies against β-actin (Cell Signaling #4970), total H3(ab1791), total H4 (ab10158), Pan-H3ac (Active Motif 39040), Pan-H4ac (Active Motif 39026), Pan-acetyllysine (Cell Signaling 9441), 4-HNE(ab46545), washed with TBST, incubated with secondary antibody and visualized using the Bio-Rad ChemiDoc MP system.

Yang L., TeSlaa T., Ng S., Nofal M., Wang L., Lan T., Zeng X., Cowan A., McBride M., Lu W., Davidson S., Liang G., Oh T.G., Downes M., Evans R., Von Hoff D., Guo J.Y., Han H, & Rabinowitz J.D. (2022). Ketogenic diet and chemotherapy combine to disrupt pancreatic cancer metabolism and growth. Med (New York, N.Y.), 3(2), 119-136.

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Examining RORγt Acetylation by p300 in Transfected 293T Cells

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293T cells were transfected at 70–80% confluency with vectors for Flag-RORγt, HA-SIRT1, and myc-p300 (pcDNA3.1) in a 1:2:4 ratio by either calcium phosphate or Lipofectamine 2000 (Invitrogen), as indicated. Total transfected DNA for each sample was normalized by adding empty vector DNA. 6–8 h after transfection, cells were washed and media was replaced followed by treatment with TSA (400 nM) for 18 h to induce p300 hyperacetylation of RORγt. Cells were harvested in p300 lysis buffer (250 mM NaCl, 0.1% NP-40, 20 mM NaH₂PO₄, pH 7.5, 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, 5 mM nicotinamide, 400 nM TSA [Sigma-Aldrich], nicotinamide [5 mM; Sigma-Aldrich], and HALT protease/phosphatase inhibitors [Thermo Fisher Scientific]). After clarification, lysates were immunoprecipitated with α-Flag (M2) conjugated-agarose (Sigma-Aldrich), washed 5 times with lysis buffer and eluted with Flag peptide (100 µg/ml). Samples were boiled in Laemmli buffer for SDS-PAGE and Western blotting, and membranes were probed with antibodies against Flag (M2, Sigma-Aldrich) and pan-acetyllysine (9441; Cell Signaling Technology).

Lim H.W., Kang S.G., Ryu J.K., Schilling B., Fei M., Lee I.S., Kehasse A., Shirakawa K., Yokoyama M., Schnölzer M., Kasler H.G., Kwon H.S., Gibson B.W., Sato H., Akassoglou K., Xiao C., Littman D.R., Ott M, & Verdin E. (2015). SIRT1 deacetylates RORγt and enhances Th17 cell generation. The Journal of Experimental Medicine, 212(5), 607-617.

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Molecular Mechanisms of SIRT6 Regulation

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Palbociclib, MK2206, AZD5363, and GSK690693 were from Selleckchem (Houston, TX). Smart pool siRNA (small interfering RNA) against human BRD4, BRD2, BRD3, and BRDT were obtained from Dharmacon (Chicago, IL). Short hairpin RNA against human FOXO3a and antibodies against Flag tag and actin were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for phospho-SirT6 (Ser338), SirT6, phospho-AKT substrate, AKT, BRD4, phospho-FOXO3a (Thr 32), FOXO3a, and Pan acetyl-lysine were purchased from Cell Signaling (Danvers, MA). Details regarding the dilutions of antibodies used were presented in Supplementary Table 1.

Liu J., Duan Z., Guo W., Zeng L., Wu Y., Chen Y., Tai F., Wang Y., Lin Y., Zhang Q., He Y., Deng J., Stewart R.L., Wang C., Lin P.C., Ghaffari S., Evers B.M., Liu S., Zhou M.M., Zhou B.P, & Shi J. (2018). Targeting the BRD4/FOXO3a/CDK6 axis sensitizes AKT inhibition in luminal breast cancer. Nature Communications, 9, 5200.

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Affinity Purification and Immunoblotting

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Whole cells were lysed by BC100 buffer (100 mM NaCl, 20 mM pH 7.3 Tris, 20% glycerol, 0.1% NP-40). The cell lysates were incubated with anti-Flag M2 (Sigma) /HA affinity gel (Roche) overnight at 4 °C. The beads were washed with BC100 6 times and eluted by Flag peptide (Sigma) at 4 °C. The elution was subjected to western blot and immunoblotted with antibodies. Flag (Sigma), HA (Pierce), α-tubulin (Santa Cruze), β-actin (Santa Cruze), GAPDH (Cell Signaling Technology), SIRT3 (Cell Signaling Technology), SIRT4 (Abiocode), PYCR1 (proteintech), pan-acetyllysine (Cell Signaling Technology /PTM Biolabs).

Chen S., Yang X., Yu M., Wang Z., Liu B., Liu M., Liu L., Ren M., Qi H., Zou J., Vucenik I., Zhu W.G, & Luo J. (2019). SIRT3 regulates cancer cell proliferation through deacetylation of PYCR1 in proline metabolism. Neoplasia (New York, N.Y.), 21(7), 665-675.

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Immunoprecipitation and Western Blotting

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Cells were disrupted by a PTY lysis buffer (35 (link)) containing the protease inhibitor cocktail (ThermoFisher Scientific). For immunoprecipitation (IP), primary antibodies were incubated with samples overnight at 4°C and followed by the addition of the protein A/G-sepharose (G&E Healthcare Life Sciences) for 2 h, precipitated by centrifugation, washed, and the immune complexes resolved by SDS-PAGE (35 (link)). The antibodies against Aurora A (AurA-N), NEDD9 (2G9), myc-tag (Santa Cruz), pan-acetyl lysine (Cell Signaling), or CTTN (4F11) were used for IP. Western blotting was conducted using standard procedures (7 (link)) with antibodies against: NEDD9 (2G9), alpha-tubulin, acetylated alpha tubulin, GAPDH, β-actin (Sigma), Aurora A (BD Biosciences), GFP (BD Bioscience), myc-tag (Santa Cruz); cortactin (Novus Biologicals), phAurora A/T288 (Cell Signaling Technology), GFP (Abcam, ab2900) and a custom made anti-HDAC6 (#7) antibody raised against a recombinant fragment of human HDAC6 (890-1048aa) (Thermo Scientific). Secondary antibodies (Jackson ImmunoResearch) and quantification methods were previously described (7 (link)).

Kozyreva V.K., McLaughlin S.L., Livengood R.H., Calkins R.A., Kelley L.C., Rajulapati A., Ice R.J., Smolkin M.B., Weed S.A, & Pugacheva E.N. (2014). “NEDD9 Regulates Actin Dynamics through Cortactin Deacetylation in an AURKA/HDAC6-dependent Manner”. Molecular cancer research : MCR, 12(5), 681-693.

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Regulation of RORγt Acetylation by SIRT1

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Total thymocytes and differentiated Th17 cells from WT and Sirt1^−/− mice were treated with TSA (2 µM) for 45 min before harvest to induce basal protein acetylation. Cells were harvested in p300 lysis buffer. After clarification, lysates were immunoprecipitated with 1.5 µg of RORγt Ab (Q31-378; BD) for 16 h at 4°C, incubated with protein A agarose beads for 6 h, washed 5 times with lysis buffer. and then eluted RORγt protein from beads with nonreducing Laemmli buffer by boiling for 3 min. Samples were subjected to Western blotting, and membranes were probed with antibodies against RORγt (B2D; eBioscience), SIRT1 (1F3; Cell Signaling Technology), and pan-acetyllysine (9441; Cell Signaling Technology)

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Subcellular Fractionation and Western Blotting

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Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with Halt protease inhibitor cocktail) to obtain whole-cell lysates or lysed using the NE-PER nuclear and cytoplasmic extraction kit to obtain cytoplasmic, nuclear, and chromatin fractions. Protein concentrations of cell fractions were determined using a BCA Assay (Thermo Fisher Scientific, 23225), and normalized among samples per experiment before analysis via western blotting using standard techniques. Proteins were visualized by chemiluminescent detection with ECL on ChemiDoc MP (Bio-Rad). Antibodies: BRD2 (Abcam, ab139690), BRD3 (Abcam, ab50818), BRD4 (Abcam, ab128874), LaminB1 (Abcam, ab16048), PARP (Cell Signaling, 9532S), GAPDH (Cell Signaling, 5174S), Strep (Qiagen, 1023944), FLAG (Sigma, F3165), Histone H4 (Cell Signaling, 13919S), pan acetyl lysine (Cell Signaling, 9441S; Abcam, ab80178), Rabbit IgG-HRP (Bethyl, A120–201P), and Mouse IgG-HRP (Bethyl, A90–516P).

Chen I.P., Longbotham J.E., McMahon S., Suryawanshi R.K., Khalid M.M., Taha T.Y., Tabata T., Hayashi J.M., Soveg F.W., Carlson-Stevermer J., Gupta M., Zhang M.Y., Lam V.L., Li Y., Yu Z., Titus E.W., Diallo A., Oki J., Holden K., Krogan N., Fujimori D.G, & Ott M. (2022). Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2. Cell Reports, 40(3), 111088.

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Immunoblotting of Sirtuin Proteins

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Western blotting was conducted as previously described [45, 46]. The primary antibodies used are as follows: anti‐Sirt3 (#5490; Cell Signaling Technology, Boston, MA, USA), anti‐Sirt4 (ab124521; Abcam, Cambridge, MA), anti‐Sirt5 (#8782; Cell Signaling Technology), anti‐α‐tubulin (Beyotime, Shanghai, China), anti‐hexokinase II (ab104836; Abcam), anti‐PDHE1α (ab168379; Abcam), anti‐SDH (ab14714; Abcam), anti‐COX‐IV (ab33985; Abcam), pan‐succinyl‐lysine (PTM‐401), pan‐malonyl‐lysine (PTM‐901), pan‐glutaryl‐lysine (PTM‐1151), pan‐acetyl‐lysine (#9441; Cell Signaling Technology) and Flag (4110‐20; Shanghai Genomics, Shanghai, China). The antibodies were purchased commercially.

Meng L., Chen D., Meng G., Lu L, & Han C. (2021). Dysregulation of the Sirt5/IDH2 axis contributes to sunitinib resistance in human renal cancer cells. FEBS Open Bio, 11(3), 921-931.

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