Fig. 4: Phosphorylation of the S279 cluster triggers the ubiquitination and subsequent degradation of N70 through the proteasome pathway.
From: A sophisticated mechanism governs Pol ζ activity in response to replication stress
a–c REV3L mRNA and protein levels were analyzed by RT-PCR (a) and western blot (b) in indicated cells, with abundance quantified via ImageJ (c). Cell types: WT, HCT116-R-3F-REV3L(het), 20 ng/mL doxycycline (DOX); 3D-Mut, HCT116-R-3F-REV3L-3D-Mut (het), 1 μg/mLDOX. d, Cells pre-treated with 100 µM cycloheximide underwent western blot analysis (left panel, asterisk marks nonspecific bands) and N70 quantitative assessment (right panel, blue for WT, orange for 3D-Mut). SEM reflects the variance over three independent experiments in (c, d). e Cells were exposed to 10 µM MG132 for 2 h. Proteins of interest were examined by western blot. A non-specific band detected by anti-REV3L-C antibody is indicated by an asterisk. f Cells were treated as described in (e). 5 × 106 cells was utilized for His-pull down experiment in the presence of 6 M guanidine hydrochloride. Flag-tagged REV3L-N70 and K48-linked ubiquitin chains in whole cell extract (WCE) and pull-downed products were monitored by western blot. (g, h) Post UV or cisplatin exposure, N70 levels were measured at specified times and doses in cells with wild-type REV3L. i UV-irradiated HCT116-R-3F-REV3L cells showed dose-dependent K48-linked polyubiquitination analyzed as in (f). j, k The abundance of N70 in cells expression wild-type and the phosphor-mimicking REV3L-3D mutant was monitored via immunoblotting. In (j) R-REV3L-3D mutant cells were cultured for indicated times in the presence of either 0.3 µM AZD6738 (ATRi) alone or ATRi in combination with 10 µM MG132. In (k) after UV irradiation, cells expressing wild-type REV3L were grown into a medium with either 0.1% DMSO (DM) or ATRi for zero (R0), one (R1), and two (R2) hours. 200 μg of whole cell extract was utilized in (b) and (e) while 100 μg was utilized for (d) (h) (j) and (k). Each experiment was independently replicated at least twice, yielding similar results each time. Source data are provided as a Source data file.
