Fig 6.

HPV8 E2 phosphorylation at S253 increases in S phase and remains high until mitosis. (A) CV-1 cell lines carrying control vector plasmid or plasmid expressing 240-255-CTD E2 protein were synchronized with thymidine and proteins extracted at 0 h (G₁/S), 3 h (S), 6 h (S/G₂), and 9 h (G₂/M) after thymidine release in RIPA extraction buffer following E2 induction for 3 h. Cell cycle stage was confirmed by flow cytometry. Mitotic cells (M) were collected by mitotic shake off at 9 h after thymidine release. Equivalent amounts of total protein were immunoprecipitated with M2 anti-FLAG antibody beads. Phosphorylated E2 protein (E2-P) was detected using phospho-RRXS motif-specific antibody, followed by reblotting for total E2 protein using rabbit anti-FLAG antibody. (B) Quantitative representation of the relative ratio of phosphorylated E2 protein to total E2 protein observed by immunoblotting in panel A. The error bars shown are a representation of the standard deviations of two experimental repeats. The asynchronous population (Asyn) has an arbitrary value of 1.