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Dynamic associations of BRD4, P-TEFb, and RNAPII with chromatin after UV treatment. Nuclear soluble fraction and chromatin fraction were isolated from UV-treated cells at different time points (0, 0.5, 1, and 2 h). Nuclear soluble fraction and chromatin fraction were lysed in SDS-loading buffer and analyzed by Western blot to determine the amount of BRD4, Cyclin T1, and CDK9 remaining in nuclear fraction and chromatin fraction, and the dynamics of total RNAPII and Ser5 and Ser2 phosphorylation from both nuclear soluble fraction and chromatin fraction. An anti-histone H3 antibody was used as a loading control for chromatin fraction. An anti-TFIIB antibody was used as the loading control for both nuclear soluble fraction and chromatin fraction.

Journal: Frontiers in Molecular Biosciences

Article Title: DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner

doi: 10.3389/fmolb.2020.618088

Figure Lengend Snippet: Dynamic associations of BRD4, P-TEFb, and RNAPII with chromatin after UV treatment. Nuclear soluble fraction and chromatin fraction were isolated from UV-treated cells at different time points (0, 0.5, 1, and 2 h). Nuclear soluble fraction and chromatin fraction were lysed in SDS-loading buffer and analyzed by Western blot to determine the amount of BRD4, Cyclin T1, and CDK9 remaining in nuclear fraction and chromatin fraction, and the dynamics of total RNAPII and Ser5 and Ser2 phosphorylation from both nuclear soluble fraction and chromatin fraction. An anti-histone H3 antibody was used as a loading control for chromatin fraction. An anti-TFIIB antibody was used as the loading control for both nuclear soluble fraction and chromatin fraction.

Article Snippet: The following antibodies were used in this study: polyclonal anti-rabbit against BRD4 (ab75898), polyclonal anti-rabbit against RNA polymerase II (Santa Cruz Biotechnologies, sc-9001), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S2) (ab5095), polyclonal anti-rabbit against CDK9 (Santa Cruz Biotechnologies, sc-8338), polyclonal anti-rabbit against Cyclin T1 (Santa Cruz Biotechnologies, sc-10750), polyclonal anti-rabbit against HEXIM1 (ab25388), polyclonal anti-rabbit against Histone H3 (ab1791), and monoclonal anti-mouse against TFIIB (ab819).

Techniques: Isolation, Western Blot, Phospho-proteomics, Control