Protocol detail

CDK9 and Cyclin T1 Western Blot

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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).

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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.

Publication 2016

Cyclin T1 ECL Western blotting system

Antibodies Cdk9 Cells Cyclin t1 Membrane Nitrocellulose Otx015 Polyacrylamide gel Protein Western blot analysis

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Protocol cited in 1 other protocol

Variable analysis

independent variables

Treatment with OTX015
Treatment with JQ1

dependent variables

Protein expression levels of CDK9, Cyclin T1, and CDK9-pT186

control variables

Culture of C11 cells
Lysis of cells on ice for 30 min
Thermal denaturation of protein extract
Loading of 50-150 mg of protein extract on a 10% polyacrylamide gel
Electroblotting onto a nitrocellulose membrane
Blocking of membrane for one hour
Incubation with antibodies (CDK9, Cyclin T1, CDK9-pT186)
Visualization of bands using the ECL Western blotting system

controls

No explicit positive or negative controls were mentioned in the protocol.

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