Fig 7.

Wild-type E2 protein has a longer half-life than phosphorylation-defective E2 protein. (A) CV-1 cell lines carrying control vector plasmid or plasmids expressing 240-255-CTD E2 protein or S253-mutated E2 protein (S253A) were either untreated or treated with 100 ng/ml CTX for 2 h, followed by E2 induction for 2 h with 4 μM CdSO₄. Thereafter, cells were treated with protein synthesis inhibitors cycloheximide (25 μM) and emetine (25 μM) for the indicated times. Cell extracts were prepared in SDS extraction buffer at the indicated time points, E2 protein was detected using M2 anti-FLAG antibody, and alpha-tubulin was detected using anti-alpha-tubulin. (B) Quantitative representation of the blot in panel A showing the percentage of E2 protein remaining plotted against time (in minutes).