Tip60

Manufactured by Santa Cruz Biotechnology

Sourced in United States, China

Tip60 is a histone acetyltransferase enzyme that plays a role in the regulation of gene expression and chromatin remodeling. It is involved in various cellular processes, including transcription, DNA repair, and apoptosis.

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8 protocols using tip60

Protein Quantification and Interactions

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Proteins were separated on 8% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and detected with primary antibody (in 3% milk in TBST or 4% PBST) against α-Actinin (B-12, SantaCruz, Cat. No. sc-166524, 1:1000), E6 (SantaCruz, Cat. No. sc-365089, 1:500), FLAG (SantaCruz, Cat. No. sc-807, 1:1000), Sp1 (SantaCruz, Cat. No. sc-59, 1:500), TIP60 (generated in the lab 1:500), Pan-acetyl lysine (SantaCruz, Cat. No. sc-8663R, 1:500), GFP-B2 (SantaCruz, Cat. No. sc-9996, 1:1000) and GFP (ROCHE, no. 11814460001 1:4000). Quantification of blots was done using Image J software. For the immunoprecipitation experiments, 4 μg of cell lysate from HeLa cells was used and immunoprecipitated using endogenous TIP60 antibody overnight. The interacting Sp1 was detected using the endogenous antibody described earlier by western blotting.

Rajagopalan D., Pandey A.K., Xiuzhen M.C., Lee K.K., Hora S., Zhang Y., Chua B.H., Kwok H.S., Bhatia S.S., Deng L.W., Tenen D.G., Kappei D, & Jha S. (2017). TIP60 represses telomerase expression by inhibiting Sp1 binding to the TERT promoter. PLoS Pathogens, 13(10), e1006681.

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Protein Expression Analysis in Cellular Extracts

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Cell extracts (30 μg) were prepared in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with a protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN) and separated on 12% SDS–PAGE gels. The expression level of the indicated proteins was assessed by probing with the following antibodies: Tfcp2l1 (OAAB09732; Aviva Systems Biology), phosphorylated Tfcp2l1 (p‐Tfcp2l1; home‐made; AbFrontier), phosphorylated threonine (p‐Thr; #9381; Cell Signaling Technology), Flag‐epitope (F3165; Sigma‐Aldrich), HA epitope (G046; Abcam), Oct‐4 (sc‐5279; Santa Cruz), Nanog (ab14959; Abcam), SOX‐2 (2683‐S; Epitomics), Klf2 (09‐820; Millipore), Klf4 (#4038; Cell Signaling Technology), cyclin A (sc‐751; Santa Cruz), cyclin B (sc‐752; Santa Cruz), cyclin D (sc‐758; Santa Cruz), cyclin E (sc‐481; Santa Cruz), CDK1 (sc‐54; Santa Cruz), histone H3 phosphorylated at serine‐10 (p‐H3S10; #9701S; Cell Signaling Technology), Hdac1 (sc‐7872; Santa Cruz), Hdac2 (sc‐7899; Santa Cruz), Hdac3 (sc‐11417; Santa Cruz), Mta1 (#5646S; Cell Signaling Technology), Mbd3 (#3896S; Cell Signaling), Ruvbl2 (#12668S; Cell Signaling Technology), Tip60 (sc‐25378; Santa Cruz), DNMAP‐1 (10411‐1‐AP; Proteintech), Trrap (#3966S; Cell Signaling Technology), pCAF (sc‐8999; Santa Cruz), and β‐actin (A5441; Sigma‐Aldrich).

Heo J., Noh B., Lee S., Lee H., Kim Y., Lim J., Ju H., Yu H.Y., Ryu C., Lee P.C., Jeong H., Oh Y., Kim K., Kim S., Son J., Hong B., Kim J.S., Cho Y.M, & Shin D. (2019). Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis. EMBO Molecular Medicine, 12(1), e10880.

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Pharmacological Inhibitors and Antibody Assays

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The pharmacological inhibitors from EMD were 200 μM epoxomycin, 40 nM MG132, 25 μM leptomycin B, 50 μM calpain inhibitor XII, 50 μM calpain inhibitor VI, and 50 μM calpeptin. The chemicals from Sigma-Aldrich were etoposide, imatinib, cisplatin, and oxaliplatin.
Antibodies against c-Myc (9E10) Sin3, Max, p53, p21, fascin, and radixin were from Santa Cruz Biotechnology. Anti-tubulin (α, β, and acetylated) and actin were from Sigma. Anti-Myc 274 and 143 were gifts from N. Ikegaki (University of Illinois). Anti-cleaved caspase 3, acetylated lysine, P-T58 Myc, MCL1, LC3A, LC3B, ATG3, ATG5, ATG7, and ATG12 were from Cell Signaling, and anti-PγH2AX and cofilin were from Abcam.
The constructs used were as follows: Myc and Myc-nick were cloned by PCR into BamHI and EcoRI sites of pBabe puro and pBabe hygro and used to prepare retrovirus. Myc and Myc-nick constructs used for transient transfections in 293T cells were cloned into pCS2⁺ vector. fascin, GCN5, Tip60, and TRRAP siRNAs were from Santa Cruz Biotechnology.
The primers used for fascin quantitative PCR were 5′-TCCACGCGCCAGGGTATGGAC-3′ and 5′-ACTTGCCCGTGTGGGTACGG-3′.

Conacci-Sorrell M., Ngouenet C., Anderson S., Brabletz T, & Eisenman R.N. (2014). Stress-induced cleavage of Myc promotes cancer cell survival. Genes & Development, 28(7), 689-707.

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Western Blot Analysis of Apoptosis Regulators

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Total cell lysates were obtained from cells homogenized in 20 mM Tris-HCl including protease inhibitor (Roche, Switzerland), left on ice for 30 min, and centrifuged for at 15,000 ×g and 4°C for 10 min. The protein concentration of each sample was determined according to the bicinchoninic acid assay. SDS-polyacrylamide gels were used to separate the denatured proteins, which were then transferred onto nitrocellulose membranes (0.2 μm). The membranes were blocked with skimmed milk (5% (w/v), dissolved in Tris-buffered saline with Tween-20 (TTBS)) for 1 h, and the antibodies against the following proteins were added: ATF, Tip60, Bax, Bcl2, β-actin (purchased from Santa Cruz (USA)), Caspase3, Foxo3a, and p-Foxo3a (acquired from Cell Signaling). The membranes were washed (thrice, 5 min each) with TTBS (0.1% (v/v)), exposed to goat anti-mouse or rabbit anti-goat IgG (1:2000 dilution) at room temperature for 1 h, then washed again (thrice, 5 min each) with TTBS. Bands were visualized via a ChemiDoc MP system (Bio-Rad, USA). Band densities were determined through ImageJ (National Institutes of Health, USA), and β-actin was used as the normalization standard for quantification.

Shen L., Lee S., Joo J.C., Hong E., Cui Z.Y., Jo E., Park S.J, & Jang H.J. (2022). Chelidonium majus Induces Apoptosis of Human Ovarian Cancer Cells via ATF3-Mediated Regulation of Foxo3a by Tip60. Journal of Microbiology and Biotechnology, 32(4), 493-503.

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Protein Characterization by Western Blotting

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Equivalent amounts of protein were suspended in protein sample buffer (50 mM Tris pH6.8, 5% SDS, 10% glycerol, 5% β-mercaptoethanol, 25mM NaF, 1mM Na₃VO₄, 5mM β-glycerophosphate, 1mM PMSF, 50 μM Leupeptin, 100 μM Pepstatin A), resolved by electrophoresis in Bis-Tris polyacrylamide gels and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were blocked in TNET (10 mM Tris pH 7.5, 50 mM NaCl, 2.5 mM EDTA pH 8.0, 0.1% tween) supplemented with 5% nonfat dry milk. Primary antibodies used in these experiments were against: HA (#12013819001, Roche), FLAG (F3165, Sigma), SMCX (NB100-55328, Novus Biologicals), EP400 (A300-541A, Bethyl Laboratories), Brd4 (Schweiger et al., 2006 (link)), TIP60 (sc-16623, Santa Cruz Biotechnology) and actin (MAB1501, Millipore). Bound antibodies were detected with horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies. For detection of actin and GAPDH, membranes were incubated with an AlexaFluor 680 conjugated goat anti-mouse IgG secondary antibody (Molecular Probes/Invitrogen) and labeled proteins visualized using a LI-COR Odyssey Infrared Imaging System.

Smith J.A., Haberstroh F.S., White E.A., Livingston D.M., DeCaprio J.A, & Howley P.M. (2014). SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter. Virology, 0, 311-321.

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Immunoprecipitation and Western Blot Analysis

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Cells were lysed for 30 min on ice in IP buffer (12 ) with protease inhibitors and TSA (Sigma). Samples were quantified with BCA assays and analyzed by western blotting. For IP, cell lysates were incubated with protein-specific antibodies or normal rabbit IgG (Santa Cruz Biotechnology, sc-2027) overnight at 4 °C, followed by incubation with protein A/G agarose beads (Millipore; IP10) for 3 h and washed in lysis buffer supplemented with protease inhibitors. Antibodies for western blotting were: Flag (Sigma; F3165), HA (Sigma; H9658), actin (Sigma; A5316), tubulin (Sigma; T6074), Acetyl-tubulin (Sigma; T7451), SIRT2 (Sigma; S8447), PKM2 (Cell Signaling; 3198), PKM2-Ac-K305 (Dr. Qun-Ying Lei; Fudan University, Shanghai, China), PCAF (Santa Cruz Biotechnology; sc-8999), Tip60 (Santa Cruz Biotechnology; sc-25378), Acetyl-lysine (Immunechem; ICP0380), and GAPDH (Millipore; MAB374).

Park S.H., Ozden O., Liu G., Song H.Y., Zhu Y., Yan Y., Zou X., Kang H.J., Jiang H., Principe D.R., Cha Y.I., Roh M., Vassilopoulos A, & Gius D. (2016). SIRT2-mediated deacetylation and tetramerization of pyruvate kinase directs glycolysis and tumor growth. Cancer research, 76(13), 3802-3812.

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Antibody Panel for Nuclear Receptor Regulation

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The following antibodies were purchased from Santa Cruz Biotechnology: anti-reptin (sc-374135), pontin (sc-393905), Tip60 (sc-166323), RORα1 (sc-26377), RORα3 (sc-38868), and β-actin (sc-8432). The following commercially available antibodies were used: anti-FLAG antibodies (Sigma, F3165), anti-lysine methyl antibodies (Abcam, ab23366), anti-dimethyl histone antibodies (Abcam, ab7766 and ab1220), and anti-RNA Polymerase II antibodies (Berkeley Antibody Company, BioLegend 920401). Anti-RORα2 antibody (target epitope is GKPPYSQKEDKEVQT-C, species: rabbit) was generated by Abmart (China) and immunized eight times with Abmart’s protocol. For Western blot assay, the dilution ratio in 5% skim milk solution was as follows: anti-reptin, pontin, Tip60, and RORα2 for 1:1000; anti-RORα1 and RORα3 for 1:500; anti-β-actin and FLAG for 1:5000.

Song H., Chu J.W., Park S.C., Im H., Park I.G., Kim H, & Lee J.M. (2020). Isoform-Specific Lysine Methylation of RORα2 by SETD7 Is Required for Association of the TIP60 Coactivator Complex in Prostate Cancer Progression. International Journal of Molecular Sciences, 21(5), 1622.

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Western Blot Analysis of Protein Modifications

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Tissue and cell lysates were prepared with RIPA lysis buffer (Beyotime, P0013B), and the protein concentrations were determined using a BCA assay kit (Beyotime, P0012S). The same amount of protein (30 μg) was separated by 8–12% SDS-PAGE and transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk, followed by incubation with primary antibodies overnight at 4 °C and secondary antibodies for 1 h. The signals were detected using an ECL kit (Advansta, CA, USA) and quantified with ImageJ software. The following primary antibodies were used: Pan-Kla (PTM-1401, diluted 1:1000); YY1-K183la (PTM, diluted 1:500); YY1 (Cell Signaling, 46395S, diluted 1:1000); SIRT1 (Abcam, ab7343, diluted 1:1000); PCAF (Abcam, ab12188, diluted 1:1000); FGF2 (Santa Cruz, sc74412, diluted 1:500); p300 (Santa Cruz, sc48343, diluted 1:500); HDAC6 (Santa Cruz, sc28386, diluted 1:500); TIP60 (Santa Cruz, sc166323, diluted 1:500); β-Actin (Proteintech, 20,536–1-AP, diluted 1:3000); VeriBlot for IP Detection Reagent (HRP) (Abcam, ab131366, diluted 1:2000), a special secondary antibody, was used for IB after IP.

Wang X., Fan W., Li N., Ma Y., Yao M., Wang G., He S., Li W., Tan J., Lu Q, & Hou S. (2023). YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2. Genome Biology, 24, 87.

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