Acetylated lysine

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Acetylated lysine is a post-translational modification of the amino acid lysine, where an acetyl group is added to the lysine residue. This modification plays a key role in regulating various cellular processes, such as gene expression, protein function, and signaling pathways.

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Total acetylated-lysine Anti-acetylated-lysine (Ac-K2-100) Anti-acetylated lysine (Lys-Ac) antibody Antibodies against acetylated lysine Acetylated-lysine antibody Acetylated-Lysine Antibody (9441) Pan-Acetylated lysine Pan-acetylated lysine antibody Acetylated-lysine (#9441 α-Acetylated Lysine

91 protocols using acetylated lysine

Elucidating Ovarian Cancer Metabolic Pathways

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Ovarian cancer cell lines (A2780, A2780/DDP, COC1, COC1/DDP) were purchased from Zhejiang Chinese Medical University. The following reagents were used: phosphatase inhibitors (Biyuntian, S1873); CCK8 detection kit (Beyotime, C0037); DATS and cisplatin (Source leaf, B25320, and B24462); annexin VFITC kit (Nanjing KGI Bio, KGA108); glucose test kit (Nanjing built, F006–11); ROS activity detection kit (Nanjing built, E004–11); glutamine and glutamate determination kit (Sigma Aldrich, GLN1); and NAC, OM, AMPK inhibitor compound C, and AMPK activator AICAR (MCE, HYB0215, HYN6782, HY13418, and HY‐13417). The following antibodies were used: pAMPK (Bioss, bs4002R); AMPK, SIRT1, and PGC1α (Affinity, DF2656, DF6033, and AF5395); acetylated‐lysine (Cell Signaling Technology, 9441); and OXPHOS (Abcam, ab110411). All other reagents were purchased from Sigma Aldrich.

Wang Z., Yan Y., Lou Y., Huang X., Liu L., Weng Z., Cui Y., Wu X., Cai H., Chen X, & Ji Y. (2022). Diallyl trisulfide alleviates chemotherapy sensitivity of ovarian cancer via the AMPK/SIRT1/PGC1α pathway. Cancer Science, 114(2), 357-369.

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Multifaceted Analysis of Protein Acetylation

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Proteins were extracted with RIPA buffer (50 mM Tris-HCL [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with NaF, NaVO₄, and Protease Inhibitor Cocktail (Cat.#P2714). Proteins were then separated in SDS-PAGE and were transferred to a nitrocellulose membrane, which was blotted with antibodies against Parkin (Cat.#2132), LC3 (Cat.#.2775), acetylated lysine (Cat#.9441) (all from Cell Signaling), cyclophyllin D (CypD, sc-137216; Santa Cruz), or Erk (SC-93; Santa Cruz). Protein bands were visualized using horseradish peroxidase-conjugated secondary antibodies and Supersignal West Femto Maximum Sensitivity Substrate (Cat.#34095; ThermoFisher). To determine acetylation levels of CypD, equal amounts of cell lysates were incubated with anti-CypD antibody overnight at 4°C, and were further incubated with protein A/G plus-agarose beads (sc-2003; Santa Cruz) for 2 h at 4°C. After washing, beads were precipitated, and proteins were processed for western blot analysis using anti-acetylated-lysine antibody (Cat.#9441; Cell Signaling). To visualize levels of CypD in the blot, the filter was stripped off the anti-acetylated-lysine antibody using Restore™ Plus stripping buffer (Cat.#46430; Thermo Scientific), and was then probed with anti-CypD antibody.

Song S.B., Jang S.Y., Kang H.T., Wei B., Jeoun U.W., Yoon G.S, & Hwang E.S. (2017). Modulation of Mitochondrial Membrane Potential and ROS Generation by Nicotinamide in a Manner Independent of SIRT1 and Mitophagy. Molecules and Cells, 40(7), 503-514.

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Mitochondrial Protein Characterization

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Mitochondrial fractions were resolved by SDS–PAGE using 10 or 12% gels and transferred to nitrocellulose membranes. Detection of specific proteins was achieved using the following antibodies and dilutions: NDUFS3 (1:5000), UQCRC2 (1:5000), and ATPB (1:5000; Mitosciences, Eugene, OR, USA); Mfn‐2 (1:5000), Drp‐1 (1:5000), acetylated lysine (1:1000), and Sirt3 (1:2000; Cell Signaling, Denvers, MA, USA); cyclophilin D (1:5000) and Hsp60 (1:5000; Abcam, Cambridge, UK).

Amigo I., Menezes‐Filho S.L., Luévano‐Martínez L.A., Chausse B, & Kowaltowski A.J. (2016). Caloric restriction increases brain mitochondrial calcium retention capacity and protects against excitotoxicity. Aging Cell, 16(1), 73-81.

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Quantitative Analysis of Protein Expression

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RNA isolation, cDNA synthesis, and quantitative PCR with the indicated primers (Supplementary Table S3) as well as Western blotting were performed as described^{28 (link)}. The following antibodies were used: NS3, ab13830, CORE, ab2740, HDAC9, and ab18970 (Abcam); FoxO1, 9454, PEPCK, 12940, GAPDH, 2118, ATF2, 9226, Acetylated-Lysine, 9681, PGC-1α, and 2178 (Cell Signalling Technology); and Acetyl-FoxO1, sc49437, PGC-1α, and sc-5815 (Santa Cruz Biotechnology).

Chen J., Zhang Z., Wang N., Guo M., Chi X., Pan Y., Jiang J., Niu J., Ksimu S., Li J.Z., Chen X, & Wang Q. (2017). Role of HDAC9-FoxO1 Axis in the Transcriptional Program Associated with Hepatic Gluconeogenesis. Scientific Reports, 7, 6102.

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Mitochondrial Dynamics Regulation in Toxicity

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Isoniazid and SB203580 (SB) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Mdivi-1, an inhibitor of DRP1, was purchased from Selleck Chemicals (Houston, TX, United States). Tetramethyl rhodamine methyl ester (TMRM), MitoTracker Deep Red FM, Hoechst 33342 and Lipofectamine RNAiMAX were obtained from Invitrogen (Grand Island, NY, United States). p38 MAPK-siRNA, a silencer negative control siRNA, and antibodies against p38 MAPK, phospho-p38 MAPK, NRF1, COX IV, cytochrome c, caspase 9, caspase 3, MFN2, DRP1, acetylated lysine, p-MAPKAPK-2, MAPKAPK-2 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against SIRT1 and Bax were obtained from Abcam (Abcam, Cambridge, United Kingdom). PGC1α antibody and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All other chemicals were of analytical grade.

Zhang T., Ikejima T., Li L., Wu R., Yuan X., Zhao J., Wang Y, & Peng S. (2017). Impairment of Mitochondrial Biogenesis and Dynamics Involved in Isoniazid-Induced Apoptosis of HepG2 Cells Was Alleviated by p38 MAPK Pathway. Frontiers in Pharmacology, 8, 753.

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Western Blot Analysis of Lipid Regulatory Proteins

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Cell lysates (30–50 μg protein) were separated on 10–12% tricine gels using a Mini-Protean II cell (Bio-Rad lab, Hercules, CA) system at constant amperage (30 mA per gel) for about 3 hrs. Proteins were then transferred onto PVDF membranes at constant voltage (90 V) for 1.5 hrs. Blots were stained with Ponceau S to confirm uniform protein loading (Aldridge et al., 2008 ; Willenborg et al., 2005 (link)) before blocking in 5% BSA in TBST (10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.05% Tween-20) for 1 hr. Blots were incubated with specific poly- or monoclonal antibodies overnight and were developed with IRDye 800CW (LI-COR) or IRDye 680RD (LI-COR) secondary antibodies. To visualize the bands of interest, blots were scanned using the LI-COR Odyssey imaging system (Lincoln, NE). Protein bands were quantitated by densitometric analysis after image acquisition using NIH Scion Image to obtain relative protein levels expressed as integrated density. All values were normalized to β-actin expression or PonceauS staining. Antibodies were purchased or obtained from the following sources; Total-Plin5 (Progen; Heudelberg, Germany), Histone H3, SIRT1, Acetylated Lysine (Cell Signaling Technologies; Danvers, MA), PGC-1α (MilliporeSigma; Burlington, MA), phospho-PLIN5 (NeoBioLab targeting; Cys-LARRGRRW(pS)VELK), PLIN2 [Barbara Atshaves developed in (Atshaves et al., 1999 (link))].

Najt C.P., Khan S.A., Heden T.D., Witthuhn B.A., Perez M., Heier J.L., Mead L.E., Franklin M.P., Karanja K.K., Graham M.J., Mashek M.T., Bernlohr D.A., Parker L., Chow L.S, & Mashek D.G. (2019). Lipid droplet-derived monounsaturated fatty acids traffic via PLIN5 to allosterically activate SIRT1. Molecular cell, 77(4), 810-824.e8.

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AMPK-p53 Signaling Modulation in Cell Assays

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Cell culture reagents were purchased from GE and fetal bovine serum (FBS) was purchased from PAN-Biotech; DTT was purchased from Millipore (Massachusetts, USA); Compound C, AICAR, Resveratrol, Suramin, SNAP, and ODQ were purchased from Sigma-Aldrich; N6022, Pifithrin-α, VO-Ohpic trihydrate, AS1842856, and MK 2206 were from MCE. Hydrogen peroxide (H₂O₂) was from Sinopharm Chemical Reagent; 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sangon Biotech; Lipofectamine® 3000 and Attractene were from Thermo Fisher Scientific and Qiagen, respectively. Fluor-de-Lys SIRT1 fluorometric drug discovery assay kit was from Enzo Life Sciences; Hydrogen Peroxide Assay Kit, Nitric Oxide Assay Kit, RIPA lysis buffer, Bicinchoninic Acid assay Kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime; Super ECL Prime was from US Everbright®Inc. The mRNA extraction kit was from Bioteke. Protein A Magnetic Beads and the Muse Count & Viability Assay Kit were purchased from Millipore. Anti-Flag M2 Magnetic Beads was from Sigma-Aldrich. Antibodies for phospho- and total-AMPK, acetyl- and total-p53, SIRT1, Cleaved- and total-Caspase-3, FOXO1, PTEN, β-actin, and acetylated-Lysine were from Cell Signaling Technology; antibodies for P21 was from Abcam; antibodies for Prdx2 was from Santa Cruz Biotechnology.

Zhang Y., Sun C., Xiao G., Shan H., Tang L., Yi Y., Yu W, & Gu Y. (2019). S-nitrosylation of the Peroxiredoxin-2 promotes S-nitrosoglutathione-mediated lung cancer cells apoptosis via AMPK-SIRT1 pathway. Cell Death & Disease, 10(5), 329.

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Cytarabine and Danorubicin Potentiation

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Cytarabine (Ara‐C) and danorubicin were purchased from Sigma‐Aldrich (St. Louis, MI, USA). PTL and 3‐(1H‐1,2,3‐triazol‐4‐yl) pyridine (3‐TYP) were from Selleckchem (Houston, TX, USA). All compounds, except for in vivo studies, were reconstituted in dimethlysulfoxide, stored at 100‐mmol/l stock concentrations at −80°C, and used at the indicated doses suggested by the vendor. Flow cytometry antibodies, Alexa Fluor 647 Rabbit Anti‐Active caspase 3, PE‐Cy7 Mouse Anti‐Human CD38, APC‐H7 Mouse Anti‐Human CD45, and APC Mouse Anti‐Human CD34 were purchased from BD Pharmingen (San Jose, CA, USA). Immunoblotting antibodies, cleaved caspase 9, MCL1, BCL2, BAD, BAX, acetylated lysine and SIRT3 were purchased from Cell Signalling Technology (Danvers, MA, USA). Acetylated SOD2 and SOD2 antibodies were purchased from Abcam. ATPA (51) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Ma J., Liu B., Yu D., Zuo Y., Cai R., Yang J, & Cheng J. (2019). SIRT3 deacetylase activity confers chemoresistance in AML via regulation of mitochondrial oxidative phosphorylation. British Journal of Haematology, 187(1), 49-64.

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Molecular Mechanisms of Cdh1, Cdc20, and APC in Cell Cycle Regulation

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The SIRT1 and AROS constructs used in this study were previously described^{32 (link)}. Full-length Cdh1, Cdc20, APC2, and APC11 were amplified via polymerase chain reaction (PCR) and subcloned into the plasmids Flag, Myc-tagged pcDNA3 (Thermo Fisher Scientific), pEGFP-C3 (BD Biosciences) and pGEX4T-1 (GE Healthcare). The antibodies used included the following: SIRT1 (Santa Cruz Biotechnology, sc15404), AROS (Santa Cruz Biotechnology, sc-86209; Abcam, ab201091), Cdh1 (Abcam, ab3242), Cdc20 (Santa Cruz Biotechnology, sc13162), Cyclin B1 (Santa Cruz Biotechnology, sc245), APC2 (Thermo Fisher Scientific, RB-067), APC11 (Abcam, ab154546), p53 (Santa Cruz Biotechnology, sc-126), p21 (Santa Cruz Biotechnology, sc-6246), p16 (Calbiochem, NA29), green fluorescent protein (GFP; Santa Cruz Biotechnology, sc-8334), polyubiquitinated conjugates (poly-Ub; Enzo Life Science, BML-PW8805-0500), acetylated-lysine (Cell Signaling Technology, 9814), hemagglutinin (HA; Merck Millipore, 05-904), Myc (Merck Millipore, 05-724), Flag M2 (Sigma‒Aldrich, F1804), LSD1 (Abcam, ab17721), H3K9me2 (Abcam, ab1120), H3K9ac (Abcam, ab12179), α-SMA-Cy3 (Sigma, C6198), collagen type I (Abcam, ab34710), elastin (Abcam, ab21600), fibronectin (Abcam, ab2413), and β-actin (Santa Cruz Biotechnology, sc47778).

Lee S.H., Yang J.H., Park U.H., Choi H., Kim Y.S., Yoon B.E., Han H.J., Kim H.T., Um S.J, & Kim E.J. (2023). SIRT1 ubiquitination is regulated by opposing activities of APC/C-Cdh1 and AROS during stress-induced premature senescence. Experimental & Molecular Medicine, 55(6), 1232-1246.

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Immunoprecipitation Assay of IRS2 Modifications

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IRS2 protein modifications were examined with an immunoprecipitation assay. Briefly, to block nonspecific background, protein G agarose beads (GE Healthcare), at a final concentration of 5% (v/v), were incubated with 500 μg total lysate from MIN6 cells in lysate buffer at 4°C for 2 h. The resulting mixture was centrifuged at 5000 rpm for 3 min, and the harvested supernatant was incubated with 4 μg IRS2 antibody with gentle agitation at 4°C overnight. Protein G beads were then added to a final concentration of 2.5% (v/v). The mixture was further incubated for 1 h at 4°C with gentle agitation and centrifuged at 5000 rpm for 3 min. The supernatant was mixed with SDS sample buffer and subjected to SDS-PAGE. The harvested beads were washed 3 times with TBS-T, re-suspended in SDS sample buffer, heat denatured, and subjected to SDS-PAGE. The proteins resolved by SDS-PAGE were profiled by immunoblot analysis using antibodies against IRS2, acetylated-lysine, phospho-tyrosine (Cell Signaling), and β-actin (Sigma-Aldrich) for definitive identification and quantification.

Kawada Y., Asahara S.I., Sugiura Y., Sato A., Furubayashi A., Kawamura M., Bartolome A., Terashi-Suzuki E., Takai T., Kanno A., Koyanagi-Kimura M., Matsuda T., Hashimoto N, & Kido Y. (2017). Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells. PLoS ONE, 12(9), e0184435.

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