Fig. 2

OTUD6A interacts with CDC6. a, Representative immunofluorescence images of endogenous OTUD6A (red) and CDC6 (green) in the indicated cells are shown. Scale bars, 20 μm. b, Pearson’s coefficient analysis was used to analyse the colocalization of OTUD6A and CDC6. c, 293 cell lysates were prepared and subjected to IP with control IgG or an anti-OTUD6A antibody. The immunoprecipitates were analysed by Western blotting. d, 293 cell lysates were prepared and subjected to IP with control IgG or an anti-CDC6 antibody. The immunoprecipitates were analysed by Western blotting. e, The indicated OTUD6A constructs were cotransfected with HA-CDC6 into 293 cells for 24 h. Whole-cell lysates were prepared and subjected to IP with an anti-Flag antibody. The immunoprecipitates were analysed by Western blotting. f, Representative confocal images of bimolecular fluorescent complimentary (BiFC) experiment are shown. White arrows represent that OTUD6A interacts with CDC6. Scale bars, 50 μm. g, A GST pull-down assay was performed to indicate the direct interaction between OTUD6A and CDC6. CBB staining, Coomassie brilliant blue staining. h, HeLa cells were synchronized at the G1/S boundary using a double-thymidine block. Whole-cell lysates were collected at the indicated time points after release into fresh medium and subjected to IP with an anti-CDC6 antibody. The immunoprecipitates were analysed by Western blotting. i, The intensities of the CDC6-bound OTUD6A (IP) and total CDC6 (IB) bands were quantified. j, 293 cells transfected with the indicated vectors were treated with HU (2.5 mM) or DMSO for 24 h. Whole-cell lysates were prepared and subjected to IP with an anti-Flag antibody. The immunoprecipitates were analysed by Western blotting