Fig. 7: DPC repair is independent of Pol II degradation.
From: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation
a, Top: scheme of experiment. Bottom: relative GFP–RPB1 protein levels measured by flow cytometry in the presence of 100 µM cycloheximide after a pulse of 1 mM FA for 30 min. RFI of GFP were normalized to mock-treated levels and set to 1. Bars represent the mean fluorescence ± s.e.m. from three independent experiments. Unpaired two-tailed t-test. b, Chromatin-bound elongating Pol II as determined by immunoblotting with the indicated antibodies. MRC-5 cells were treated with 1 mM FA for 30 min and collected at the indicated times. SSRP1 was used as the loading control. This experiment was performed three times with similar results. c, Quantification of pSer2-modified RPB1 levels as shown in b. Values indicate the average integrated density ± s.e.m. from three independent experiments. Unpaired two-tailed t-test. d, Relative survival of WT, CSB KO or K1268R mutated RPB1 HeLa cells treated with the indicated doses of FA for 1 h. Values represent the mean ± s.e.m. from three independent experiments. Unpaired two-tailed t-test at 1.5 mM FA. e, Quantification of recovery of transcription in WT, CSB KO or K1268R mutated RPB1 HeLa cells after a 30 min pulse of 1 mM FA. After FA washout, cells were left to recover for the indicated times, including a 30 min pulse labelling with EU. RFI of EU were normalized to mock-treated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 1,311, 1,169, 1,162, 1,224, 1,439, 1,360, 1,236, 1,386, 973, 1,286, 1,310 and 1,107 cells from 3 independent experiments. Source numerical data (a,c–e) and unprocessed blots (b) are available in the source data.
