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. 2012 Oct 30;109(46):18797–18802. doi: 10.1073/pnas.1207483109

Fig. 4.

Fig. 4.

The level of phospho-TFIIB ser65 is reduced upon treatment with DNA-damaging agents. (A) qPCR analysis of AREG, EREG, p21, and BAX gene expression was performed at different time points of 5 µg/mL DoxR treatment in HCT116WT cells (Upper). TFIIB was immunoprecipitated from HCT116WT cells after different time points of 5 µg/mL DoxR treatment and immunoblotted using anti-pS65-TFIIB and anti-TFIIB antibodies (Lower). (B) ChIP analysis was carried out with anti-pS65-TFIIB and anti-TFIIB antibodies in HCT116WT cells treated with 5 µg/mL DoxR after different time points. The normalized level of phospho-S65TFIIB (over TFIIB ChIP signal) at the AREG, p21, and BAX promoters is plotted as fold enrichment relative to 18S DNA. (C) ChIP analysis was also carried out with anti-p53 and anti-RNAPII antibodies in HCT116WT cells treated with 5 µg/mL DoxR after 3 h and compared with untreated cells. The levels of p53 and RNAPII at the upstream promoter sequence (p53-binding site [bs] at −2 and −1 Kb) and core promoter region of p21 and core promoter of AREG is plotted as fold enrichment relative to 18S DNA. (D) TFIIB was immunoprecipitated from HCT116WT cells after 8 h of 10 μM Nutlin3 or control DMSO treatment and immunoblotted using anti-pS65-TFIIB and anti-TFIIB antibodies (Upper). HCT116WT cells were treated with the indicated concentrations of Nutlin3 for 8 h and immunoblotted with anti-p53 antibody and β-tubulin antibodies (Lower). (E) qPCR analysis of the indicated mRNAs was performed after 8 h of 10 μM Nutlin3 treatment in HCT116WT cells (F) ChIP analysis was carried out with anti-pS65-TFIIB and anti-TFIIB antibodies in HCT116WT cells treated with 10 μM Nutlin3 for 8 h. The normalized level of phospho-S65TFIIB (over TFIIB ChIP signal) at the AREG and p21 promoters is plotted as fold enrichment relative to 18S DNA. (G) As in F, Chip assay was done with anti-p53 antibody to analyze its occupancy at the indicated regions of p21 promoter. Error bars denote SD.