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Cyclin t1

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Cyclin T1 is a protein involved in the regulation of the cell cycle. It is a subunit of the positive transcription elongation factor b (P-TEFb) complex, which plays a critical role in the regulation of RNA polymerase II-dependent transcription.

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Lab products found in correlation

β actin, by Cell Signaling Technology (2 mentions) Full length caspase 3, by Cell Signaling Technology (1 mentions) Tubulin, by Cell Signaling Technology (1 mentions) Mcl 1, by Santa Cruz Biotechnology (1 mentions) Phospho ctd ser2, by Merck Group (1 mentions) Phospho ctd ser5, by Merck Group (1 mentions) Total pol 2, by Santa Cruz Biotechnology (1 mentions) Infra red labeled antibodies, by LI COR (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Odyssey clx imager, by LI COR (1 mentions) Protease inhibitor, by Roche (1 mentions) Phosstop phosphatase inhibitor, by Roche (1 mentions) Anti rabbit igg, by Thermo Fisher Scientific (1 mentions) H3k79me1, by Cell Signaling Technology (1 mentions) H3k79me2, by Cell Signaling Technology (1 mentions) Histone h3, by Cell Signaling Technology (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Protease inhibitor cocktail, by Abcam (1 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Phosphorylated nf κb p65 ser536, by Cell Signaling Technology (1 mentions)

4 protocols using cyclin t1

1

Protein Lysis and Western Blot Analysis in MOLT4 Cells

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MOLT4 cells were lysed in RIPA buffer (50mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.4 ± 0.2) with Protease Inhibitor (Roche), Phosstop Phosphatase Inhibitor (Roche), and 2.5 U/mL Universal Nuclease for Cell Lysis (Pierce) by incubating on ice 30 minutes. The lysates were clarified by spinning at 21,000g for 30 minutes at 4°C and the concentration of the lysate was determined using BCA protocol (Pierce). Primary antibodies in this study include: CDK9 (Cell Signaling Technology, 2316), CDK7 (Cell Signaling Technology, 2916), CDK2 (Bethyl Labs, A301-812), CDK1 (Bethyl Labs, A303-663), Cereblon (Novus Biologicals, NBP1-91810), Tubulin (Cell Signaling Technology, 3873), PARP (Cell Signaling Technology, 9542), MCL-1 (Santa Cruz Biotechnology, sc-819), Caspase-3 Full Length (Cell Signaling Technology,9668), Caspase-3 Cleaved (Cell Signaling Technology, 9661), ɣH2A.X (Cell Signaling Technology, 9718), Phospho-CTD Ser2 (Millipore, 04-1571), Phospho-CTD Ser5 (Millipore, 04-1572), Total Pol II (Santa Cruz Biotechnology, sc-899), Cyclin T1 (Cell Signaling Technology, 8744), CDK10 (Cell Signaling Technology, 36106), ENL (Cell Signaling Technology, 14893), AFF4 (Abcam, ab103586), and ELL (Cell Signaling Technology, 14468S). Secondary antibodies used were infra-red labeled antibodies (LI-COR) and blots were imaged on an Odyssey CLX imager.
Olson C.M., Jiang B., Erb M.A., Liang Y., Doctor Z.M., Zhang Z., Zhang T., Kwiatkowski N., Boukhali M., Green J.L., Haas W., Nomanbhoy T., Fischer E.S., Young R.A., Bradner J.E., Winter G.E, & Gray N.S. (2017). Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nature chemical biology, 14(2), 163-170.
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2

Compound Dose-Dependent Protein Analysis

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3 × 106 cells/well were treated with increasing concentrations of a compound for 1 day, and whole proteins were extracted. Equal amounts of proteins were separated on SDS-PAGE and transferred to PVDF membranes. The blots were probed with primary antibodies, followed by anti-rabbit IgG (Thermo Scientific) secondary antibodies. The primary antibodies against ENL (Cell signaling #14893), AF9 (Novusbio #NB100-1565), DOT1L (Cell signaling #77087), AFF4 (Abcam #ab103586), Cyclin T1 (Cell signaling #81464), H3K79me2 (Cell signaling #5427), H3K79me1 (Cell signaling #9398), Histone H3 (Cell signaling #4499), FLAG (Sigma-Aldrich #F1804), β-Actin (Cell signaling #4970) were used in this study.
Li X., Yao Y., Wu F, & Song Y. (2022). A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth. Journal of Hematology & Oncology, 15, 41.
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3

Western Blot Analysis of Cellular Proteins

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Cell pellets were washed in cold PBS and lysed with buffer containing 1% NP40 and protease inhibitor cocktail (Abcam). Whole cell lysates were obtained by centrifugation and removal of cell debris. Protein concentration of lysates was determined by the Pierce BCA Assay (ThermoFisher Scientific). Then, 15 to 20 μg of total protein was loaded onto precast 4 to 12% iBlot2 Bis-Tris gels (ThermoFisher Scientific) and allowed to run for ~90 min at 120 V. Proteins were transferred onto a polyvinylidene difluoride (PDVF) membrane using the iBlot2 Dry Blotting System (ThermoFisher Scientific). The membrane was blocked in 5% nonfat milk or bovine serum albumin (BSA) for 1 h and incubated overnight at 4 °C with primary antibodies against Cyclin T1 (Cell Signaling Technologies), p24 (Abcam), phosphorylated NF-κB p65 (Ser536) (Cell Signaling Technologies), NF-κB (Cell Signaling Technologies), and B-actin (Cell Signaling Technologies) at dilutions recommended by the manufacturer. Proteins were detected by horseradish peroxidase-conjugated secondary antibody using SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific) and imaged with an Invitrogen iBright FL1500 imaging system (ThermoFisher Scientific). Results were quantified by determining the area under peak for each band in ImageJ.
Blanco A., Coronado R.A., Arun N., Ma K., Dar R.D, & Kieffer C. (2024). Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation. Proceedings of the National Academy of Sciences of the United States of America, 121(19), e2313823121.
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4

CDK9 and Cyclin T1 Western Blot

Cited 1 time
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Western blot analysis was performed as described74 (link). C11 cells were treated with OTX015 or JQ1 for 24 h and lysed on ice for 30 min. Approximately 50–150 mg of thermally denatured protein extract was loaded on a 10% polyacrylamide gel, electroblotted onto a nitrocellulose membrane and blocked for one hour. The membrane was then incubated with CDK9, Cyclin T1 or CDK9-pT186 antibodies (Cell Signaling Technology). Bands were visualized using the ECL Western blotting system (Santa Cruz Biotechnology).
Lu P., Qu X., Shen Y., Jiang Z., Wang P., Zeng H., Ji H., Deng J., Yang X., Li X., Lu H, & Zhu H. (2016). The BET inhibitor OTX015 reactivates latent HIV-1 through P-TEFb. Scientific Reports, 6, 24100.
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