Extended Data Fig. 2. CSB-dependent ubiquitination signaling is activated in response to formaldehyde treatment.
(a) Whole cell extracts of WT, ΔCSB and POLR2AKR HeLa cells after formaldehyde treatment or UV irradiation were analysed by immunoblotting with indicated antibodies. Experiments were independently replicated twice with consistent results. (b) Volcano plot of mass spectrometry analyses illustrating the UV-induced interaction of elongating RNAPII. Chromatin fractions of HeLa WT cells after UV irradiation (UV) or untreated (UT) were subjected to immunoprecipitation with anti-RPB1 CTD phospho-Ser2 antibodies followed by mass spectrometric analysis. The plot displays the log2 fold change and the significance (-log10(P-value)) assessed by unpaired two-sided t test: permutation-based FDR < 0.05 (n = 3). Selected prospective elongating RNAPII interactors are highlighted. See Supplementary Table 4 for full results. (c) Volcano plot of mass spectrometry analyses illustrating the CSB-dependent formaldehyde-induced interaction of elongating RNAPII. Chromatin fractions of HeLa WT and ΔCSB cells after formaldehyde treatment were subjected to immunoprecipitation with anti-RPB1 CTD phospho-Ser2 antibodies followed by mass spectrometric analysis. The plot displays the log2 fold change and the significance (-log10(P-value)) assessed by unpaired two-sided t test: permutation-based FDR < 0.05 (n = 3). Selected prospective elongating RNAPII interactors are highlighted. See Supplementary Table 5 for full results. (d) Volcano plot of mass spectrometry analyses illustrating the CSB-dependent UV-induced interaction of elongating RNAPII. Chromatin fractions of HeLa WT and ΔCSB cells after UV irradiation were subjected to immunoprecipitation with anti-RPB1 CTD phospho-Ser2 antibodies followed by mass spectrometric analysis. The plot displays the log2 fold change and the significance (-log10(P-value)) assessed by a permutation-based t-test (n = 3). Selected prospective elongating RNAPII interactors are highlighted. See Supplementary Table 6 for full results. (e) Chromatin fractions of WT, ΔCSB, ΔUVSSA and POLR2AKR HeLa cells after formaldehyde treatment were subjected to immunoprecipitation with anti-RPB1 phospho-Ser2 antibodies followed by immunoblotting with indicated antibodies. * indicates non-specific bands. Experiments were independently replicated twice with consistent results. Unprocessed blots are available in source data.