Fig. 1

Transcription induces SUMO2 conjugation of PCNA at K164. a Schematic of the cell fractionation procedure used to separate proteins associated with transcriptionally active open chromatin (CB:RNA+) and proteins bound to not highly or non-transcribed DNA regions (CB:RNA−). b Western blot analysis of the indicated proteins in CB:RNA+ and CB:RNA− fractions prepared from HEK293T cells with or without 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) treatment to inhibit transcription. The post-translationally modified form of PCNA is indicated with an asterisk (*). c Cell cycle analysis (by flow cytometry) of HEK293T cells after release from nocodazole (Noc) at the indicated time points. 1 C and 2 C indicate cells containing one or two copies of each chromosome, respectively. d Western blot analysis of PCNA and SRSF1 in CB:RNA+ and CB:RNA- fractions prepared from HEK293T cells shown in c. Histone H4 was used as a loading control. e Western blot analysis using the indicated antibodies to detect His-Myc-tagged PCNA purified under denaturing conditions from the CB:RNA+ and CB:RNA− fractions of HEK293T cells expressing His-Myc-PCNA. f Western blot analysis using an α-PCNA antibody to detect SUMOylated PCNA purified using Ni-NTA under denaturing conditions from whole cell extracts (WCE, center panel) and CB:RNA+ fractions (bottom panel) of HEK293T cells transfected with an empty His vector, His-SUMO1, His-SUMO2, or His-SUMO3. Unconjugated His-SUMO was detected using α-His antibody (top). Protein bands that cross-reacted with the α-PCNA antibody are indicated with a (<). g Western blot analysis of WT and K110R, K117R, K138R, K164R, K168R, and K254R FLAG-PCNA mutants in the CB:RNA+ and CB:RNA− fractions of HEK293T cells. Blots were probed using an α-FLAG antibody