Fig. 2
SUMO2-PCNA enhances replication fork progression. a Schematic diagram of FLAG-PCNA WT, K164R (KR), and SUMO2-KR (S2-KR) fusion constructs. The location of KR mutation is shown in red. b Western blot analysis of PCNA and FLAG-PCNA WT, KR, and S2-KR using an α-PCNA antibody and an α-RNAPII A10 antibody in whole cell extracts (WCE) prepared from HEK293T cells overexpressing the indicated FLAG proteins. Histone H4 was used as a loading control. c Western blot analysis of the indicated proteins in the chromatin-bound (CB) fractions of the HEK293T cells overexpressing the indicated FLAG-PCNA proteins in b. H5 mouse monoclonal was used to detect RNAPIIo. d Representative fluorescence images of DNA fibers isolated from HEK293T cells overexpressing the indicated FLAG-PCNA proteins or transfected with an empty vector. Scale bar=10 µm. e Distributions of the replication fork processivity rates for HEK293T cells used in d overexpressing the indicated FLAG-PCNA proteins or transfected with an empty vector. Fiber length was measured based on a conversion factor of 1 µm to 2.59 kb. Each average value ± standard deviation was calculated from at least 150 DNA fibers per one representative experiment. The result was reproduced in two independent assays
