Fig. 6

Inhibiting PAD2 combined with docetaxel treatment promoted nuclear accumulation of p53 in MCF7/TamR cells, and PAD2 facilitates p53 degradation by ubiquitination. a Cellular proteins after 0.1 μM docetaxel and 25 μM cl-amidine treatment were separated into cytoplasmic and nuclear pools by fractionation methods and examined by western blot with anti-p53 antibody. Cleanliness of fractionation was determined by probing with antibodies for Histone H3 (nuclear) and GAPDH (cytoplasmic) proteins. b HEK293 cells were transfected with Flag-tagged PAD2 followed by cellular fractionation analysis. Western blotting showing that cellular p53 expression was not affected by PAD2 overexpression, but nuclear p53 expression decreased. c HEK293 cells were transfected with HA-tagged ubiquitin (HA-Ub) and Flag-tagged PAD2, followed by immunoprecipitation assay by anti-p53 antibody. The immunoprecipitates were detected by anti-HA antibody. GAPDH served as loading control