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. Author manuscript; available in PMC: 2021 Jul 12.
Published in final edited form as: Nat Cell Biol. 2021 Jun 9;23(6):608–619. doi: 10.1038/s41556-021-00692-z

Figure 6. ELOF1 is crucial for proper TC-NER complex assembly.

Figure 6.

(a) Immunoprecipitation of P-Ser2-modified Pol II in Wt and ELOF−/−A cells followed by immunoblotting for indicated proteins. Cells were harvested 1 hour after mock treatment or irradiation with 16 J/m2 UV-C. (b) Left panel: Relative immobile fraction of CSB in CSB-mScarletI KI cells transfected with indicated siRNAs directly (UV) or 5 hours after UV-C irradiation (5h UV, 4 J/m2) as determined by FRAP analysis (Extended data fig. 7e). Right panel: Relative fluorescence intensity of CSB-mScarletI in CSB-KI cells transfected with indicated siRNAs as determined by live-cell imaging. Values represent mean ± SEM and are normalized to mock-treated. siCTRL NT+UV n=30, 5h UV n=20; siELOF1#1 NT n=30, UV n=25, 5h UV n=9; siELOF1#2 NT+UV n=25, 5h UV n=18 cells analyzed across 2 independent experiments, siELOF1#1 5hr UV was performed once (c) Immunoprecipitation of P-Ser2-modified Pol II in Wt and ELOF−/−A cells 1 or 5 hours after UV-C (16 J/m2) irradiation followed by immunoblotting for indicated proteins. IgG was used as binding control. *non-specific band. (d) Same as left panel of (b) but for UVSSA-mScarletI KI cells (Extended data fig. 7f,g). siCTRL NT n=30, UV+5h UV n=21; siELOF1#1 NT n=38, UV n=34, 5h UV n=16; siELOF1#2 NT+UV n=19, 5h UV n=16 cells analyzed across 2 independent experiments. (e) Relative immobile fraction (left panel) or relative fluorescence intensity (right panel) of CSB-mScarletI in CSB-KI cells transfected with indicated siRNAs 2 hours after UV-C irradiation (4 J/m2) as determined by FRAP analysis (Extended data fig. 7h). VCPi: treatment with VCP inhibitor. Vvalues represent mean ± SEM and are normalized to mock-treated. siCTRL: NT n=30, 2h UV n=15, 2h UV+VCPi n=17; siELOF1#2: NT n=25, 2h UV n=20, 2h UV+VCPi n=22 cells analyzed across 2 independent experiments. (f) Immunoblot of chromatin fraction of indicated HCT116 Wt or ELOF1 KO cells, with re-expression of Wt of Zinc-finger mutant ELOF1, 1 hour after 12 J/m2 UV-C or mock treatment. Loading control; BRG1. Data shown in a, c, f has been performed 2 times with similar results. Numerical data and uncropped blots are provided in source data fig. 6.