Figure 6. Phosphorylation dependencies that target Cdc25A to β-TrCP.
Cells transfected with plasmids expressing WT and mutant forms of Cdc25A for 20 h were lysed and lysates were resolved by SDS-PAGE. Western blotting was performed to monitor S76- (A), S88- (B), S82- (C) and S79 (D)-phosphorylation. (E) HeLa cells transfected with plasmids encoding tagged forms of Cdc25A and β-TrCP for 20 h were lysed and resolved directly by SDS-PAGE (WCE) or were incubated first with Flag agarose to precipitate Cdc25A. Precipitates were then resolved by SDS-PAGE followed by Western blotting. (F) Cells expressing WT and mutant forms of Cdc25A were either lysed immediately (time = 0) or were incubated in the presence of 10 μg/ml cycloheximide (CHX) for the indicated times prior to processing. Lysates were resolved by SDS-PAGE and subjected to Western blotting.