Rabbit anti acetylated lysine

Manufactured by Cell Signaling Technology

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Rabbit anti-acetylated lysine is a primary antibody that binds to acetylated lysine residues in proteins. It is used for the detection and analysis of acetylated proteins in various experimental applications.

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Acetylated lysine (91 citations) Anti-acetylated-lysine (43 citations) Rabbit anti-acetylated-Lysine antibody (3 citations)

7 protocols using rabbit anti acetylated lysine

Western Blotting and Immunofluorescence Antibodies

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The following antibodies were used for Western blotting: anti-FLAG M2-peroxidase clone M2 (1:10 000; Sigma), mouse anti-FLAG M2 (1:5000; Sigma), mouse anti-HA.11 clone 16B12 (1:1000; Covance), mouse anti-myc clone 4A6 (1:1000; Merck Millipore), rabbit anti-acetylated-lysine (1:1000; Cell Signaling), mouse anti-p300 (1:1000; ab14984), rabbit anti-PCNA (1:1000; ab18197), mouse anti-α-tubulin clone B512 (1:5000; Sigma), rabbit anti-PAR (1:1000; Trevigen), rabbit anti-PARP1 (1:1000; Cell Signaling). The following antibodies were used for immunofluorescence: mouse anti-AIM-1 (1:600; BD), rabbit anti-PCNA (1:700; ab18197), mouse anti-GFP (1:500; Roche). Secondary HRP-conjugated antibodies for western blotting (Jackson ImmunoResearch) or IRDye fluorescent dye-conjugated antibodies (LI-COR) were used at 1:10000 dilution. Secondary Alexa Fluor^® antibodies for immunofluorescence (Life Technologies) were used at 1:500 dilution.

Kaufmann T., Grishkovskaya I., Polyansky A.A., Kostrhon S., Kukolj E., Olek K.M., Herbert S., Beltzung E., Mechtler K., Peterbauer T., Gotzmann J., Zhang L., Hartl M., Zagrovic B., Elsayad K., Djinovic-Carugo K, & Slade D. (2017). A novel non-canonical PIP-box mediates PARG interaction with PCNA. Nucleic Acids Research, 45(16), 9741-9759.

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Investigating CUDC907-Induced Apoptosis Signaling

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HS01 cells were seeded at 250,000 cells/well in 6-well CellBIND (Corning) plates, grown to ~80% confluency, then treated with CUDC907 for indicated times. Cells were extracted with 4% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, 10% glycerol and 100 mmol/L dithiothreitol. Antibodies were used from Cell Signaling Technology: rabbit anti-cleaved caspase 7 (1:500; #8438); rabbit anti-cleaved caspase 3 (1:1000, #9664); rabbit anti-acetylated lysine (1:1000; #9814); mouse anti-actin (1:30,000; #3700); rabbit anti-pFAK(Y397) (1:1000; #8556), mouse anti-FAK (1:500; #3285); rabbit anti-pERK (1:1000; #4370); mouse anti-ERK (1:500; #9107); rabbit anti-pAKT (T308) (1:1000; #29655); mouse anti-AKT (1:1000; #29205); rabbit anti-pAKT (S473) (1:1000; #4060); rabbit anti-YAP (1:1000; #14074); rabbit anti-pYAP (S127) (1:1000; #4911). Primary antibodies were diluted in 1:1 tris-buffered saline-0.1% Tween and Odyssey Blocking Buffer or 5% milk and incubated overnight at 4°C. Secondary antibodies were diluted in same solution and incubated for 45 minutes at room temperature. Western blots were imaged on the LI-COR Odyssey Imaging System (LI-COR Biosciences) or ChemiDoc (Bio-Rad) and quantified on ImageJ (NIH).

Huegel J., Dinh C.T., Martinelli M., Bracho O., Rosario R., Hardin H., Estivill M., Griswold A., Gultekin S., Liu X.Z, & Fernandez-Valle C. (2022). CUDC907, a dual phosphoinositide-3 kinase/histone deacetylase inhibitor, promotes apoptosis of NF2 Schwannoma cells. Oncotarget, 13, 890-904.

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Western Blot and Immunofluorescence Antibody Protocol

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The following antibodies were used for western blotting: rabbit anti-SIRT2 (1:1000; Sigma #S8447 or Cell Signaling #12650), rabbit anti-ANKLE2 (1:500; Atlas Antibodies #HPA003602), goat anti-RTN4 (1:1000; Nogo N18, Santa Cruz Biotechnology #sc-11027), anti-FLAG M2-peroxidase clone M2 (1:10,000; Sigma #A8592), streptavidin–HRP (1:1000; GE Healthcare #RPN1231), mouse anti-HA.11 clone 16B12 (1:1000; Covance #MMS-101R), mouse anti-Myc clone 4A6 (1:1000; Merck Millipore #05-724), rabbit anti-acetylated-lysine (1:1000; Cell Signaling #9441), mouse anti-α-tubulin clone B512 (1:5000; Sigma #T5168), mouse anti-His (1:5000; GE Healthcare #27471001), mouse anti-GFP (1:1000; Roche #11814460001). The following antibodies were used for immunofluorescence: rabbit anti-SIRT2 (1:200; Sigma #S8447), mouse anti-Myc clone 4A6 (1:500; Merck Millipore #05-724), streptavidin–Alexa-Fluor-568 (1:500; Life Technologies #S11226), rabbit anti-calnexin (1:200; a gift from Erwin Ivessa; Medical University of Vienna, Austria), mouse anti-lamin A/C (1:100; Millipore #MAB3211), mouse anti-acetylated tubulin (1:1000; Sigma #T7451). Secondary HRP-conjugated antibodies for western blotting (Jackson ImmunoResearch) were used at 1:10,000 dilution. Secondary Alexa-Fluor^®-conjugated antibodies for immunofluorescence (Life Technologies) were used at 1:500 dilution.

Kaufmann T., Kukolj E., Brachner A., Beltzung E., Bruno M., Kostrhon S., Opravil S., Hudecz O., Mechtler K., Warren G, & Slade D. (2016). SIRT2 regulates nuclear envelope reassembly through ANKLE2 deacetylation. Journal of Cell Science, 129(24), 4607-4621.

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Establishment of SIRT1 Inhibition Model

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BAI (Figure 1(a); 98% purity, CAS number 21967-41-9) was bought from Tauto Biotech (Shanghai, China). LPS from Escherichia coli O111:B4 and protease and phosphatase inhibitor tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). EX-527, also known as Selisistat, was purchased from Selleck (Houston, TX, USA). Rabbit anti-SIRT1, rabbit anti-HMGB1, rabbit antiacetylated lysine, and mouse anti-GFAP were from Cell Signaling Technology (Beverly, MA, USA); mouse anti-TNF-α and mouse anti-HMGB1 were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-IL-1β, goat antimouse IgG H&L (Alexa Fluor® 594), and goat antirabbit IgG H&L (Alexa Fluor® 594/488) were from Abcam (Cambridge, MA, USA); rabbit anti-Iba-1 was from Wako (Rosemont, IL, USA); mouse anti-LaminB1, mouse anti-GAPDH, and mouse anti-β-actin were from Proteintech Group (Wuhan, China). Protein A/G-coupled magnetic beads were from Invitrogen (Carlsbad, CA, USA).

Li Y., Liu T., Li Y., Han D., Hong J., Yang N., He J., Peng R., Mi X., Kuang C., Zhou Y., Han Y., Shi C., Li Z, & Guo X. (2020). Baicalin Ameliorates Cognitive Impairment and Protects Microglia from LPS-Induced Neuroinflammation via the SIRT1/HMGB1 Pathway. Oxidative Medicine and Cellular Longevity, 2020, 4751349.

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Immunoblot Analysis of Protein Acetylation

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Whole-cell extracts were prepared and immunoblots performed on 30 µg of extract using standard procedures (Thoms et al., 2010 (link)). Primary antibodies were: rabbit anti-p65 (C-20) (Santa Cruz), mouse anti-p65 (Santa Cruz Biotechnology), rabbit anti-p65 (acetyl K310) (Abcam), mouse anti-His (generated in house), mouse anti-p300 (BD Pharmingen), rabbit anti-acetylated lysine (Cell Signaling), mouse anti-COMMD1 (Abnova), anti-XIAP (Santa Cruz) and anti-GST–HRP (Abcam) antibodies. Actin (Amersham) controlled for protein loading.

O'Hara A., Simpson J., Morin P., Loveridge C.J., Williams A.C., Novo S.M, & Stark L.A. (2014). p300-mediated acetylation of COMMD1 regulates its stability, and the ubiquitylation and nucleolar translocation of the RelA NF-κB subunit. Journal of Cell Science, 127(17), 3659-3665.

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Antibody Panel for Cellular Analysis

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The following antibodies were used in this study: rabbit anti-CTCF (active motif, 61,311), mouse anti-Flag (Sigma, F1804), rabbit anti-acetylated lysine (Cell Signaling Technology, 9441S), mouse anti-β-ACTIN (Abcam, ab8226), rabbit anti-HDAC6 (Proteintech, 12,834–1-AP), rabbit anti-CBP (Cell Signaling Technology, 7389S), mouse anti-MOF (Boster, A02757), rabbit anti-H3K27ac (Active Motif, 39,133), anti-OCT4 (Santa Cruz Biotechnology, sc-5279), anti-SOX2 (Abcam, ab79351), mouse anti-cTnT (Thermo, MA5-12,960), rabbit IgG (Santa Cruz Biotechnology, sc-2027), and anti-6 × HIS (Abcam, ab18184).

Gong S., Hu G., Guo R., Zhang J., Yang Y., Ji B., Li G, & Yao H. (2022). CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells. Cell Regeneration, 11, 34.

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Detecting NNMT and PGC-1α Acetylation

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NNMT was detected using a chicken anti-NNMT antibody from GenWay Biotech or the rabbit anti-NNMT antibody kindly provided by R. Weinshilboum^{34 (link)}. Goat anti-UCP1 (M-17) and rabbit anti-PGC1α (H-300) were from Santa Cruz Biotechnology. Rabbit anti-acetylated lysine was from Cell Signaling Technology. For detecting PGC-1α acetylation, PGC-1α was immunoprecipitated from liver or cultured-cell lysates with anti-PGC-1α. The precipitated protein was subjected to western blot analysis using anti-acetylated lysine.

Kraus D., Yang Q., Kong D., Banks A.S., Zhang L., Rodgers J.T., Pirinen E., Pulinilkunnil T.C., Gong F., Wang Y.C., Cen Y., Sauve A.A., Asara J.M., Peroni O.D., Monia B.P., Bhanot S., Alhonen L., Puigserver P, & Kahn B.B. (2014). Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature, 508(7495), 258-262.

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