Fig. 7
RECQ5 promotes SUMO2 conjugation of PCNA. a Western blot analysis of RECQ5 in whole cell extracts (WCE) prepared from control or RECQ5 siRNA knockdown HEK293T cells treated with or without camptothecin (CPT, 5 µM). Actin was used as a loading control. b Western blot analysis of PCNA in the CB:RNA+ and CB:RNA− fractions prepared from HEK293T cells with or without CPT treatment from a. c Western blot analysis of the indicated proteins in CB:RNA+ and CB:RNA− fractions prepared from HEK293T cells with or without exogenous overexpression of FLAG-RECQ5. MCM7 was used as a loading control. d Western blot analysis of the indicated proteins in the input of the CB:RNA+ and CB:RNA− fractions with or without exogenous overexpression of FLAG-RECQ5 visualized with short and long exposure times (top) and in the FLAG-RECQ5 complex purified from the CB:RNA+ and CB:RNA− fractions shown above (bottom). e Amino acid sequences of the PCNA-Interacting-Protein (PIP) and PIP-like (PIP-L) motifs found in human RECQ5 protein are shown in bold. f Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells with or without exogenous overexpression of FLAG-RECQ5 WT and mutant proteins. g Western blot analysis of the indicated proteins in the chromatin-bound (CB) fractions prepared from HEK293T cells from f. h Western blot analysis of the indicated proteins in the FLAG-RECQ5 complexes purified from CB fractions shown in g. i Western blot analysis of the indicated proteins in WCE prepared from control or RECQ5 siRNA knockdown HEK293T cells with exogenous overexpression of indicated FLAG-tagged PCNA constructs or CAF1 or treated with an empty vector (V). j Quantification of γH2AX-positive cells indicated in i. Each value in the graph represents the average value ± standard deviation (n > 200) per one representative experiment. Only cells with 5 or more γH2AX foci were counted as positive cells. p values equal to or less than 0.05 are indicated with an asterisk (*).The result was reproduced in two independent assays
