Fig. 2.

PR Ser81 is phosphorylated by endogenous ck2. (A and B) HeLa-PR (A) and T47D-YB (B) cells were serum starved for 18 h. Cells were then pretreated with increasing doses of TBB (1 to 100 μM), DMAT (1 to 100 μM), or DMSO (vehicle) for 30 min, followed by 10 nM R5020 for 30 min. Alternatively, cells were treated with R5020 for 30 min or vehicle (EtOH) with no pretreatment. Lysates were analyzed by Western blotting using p-S81, PR, and Erk1/2 antibodies. (C) HeLa-PR cells were starved for 18 h in serum-free medium. Cells were then pretreated (30 min) with TBB (10 μM), DMAT (10 μM), PP2 (10 μM), Roscovitine (100 μM), U0126 (10 μM), or vehicle (DMSO) or left untreated. Following kinase inhibitor pretreatments, cells were treated with 10 nM R5020 or vehicle (EtOH) for 30 min. Lysates were analyzed by Western blotting as described for panel A. (D) Left: T47D-YB cells were serum starved for 18 h and treated with EtOH or 10 nM R5020 for 60 min (left two lanes). Alternatively, cells were treated sequentially as follows: 18 h with thymidine (2.5 μg/ml) or vehicle (PBS), iMEM plus 5% DCC for 7 h, iMEM-5% DCC-mimosine (50 μg/ml; G1/S Sync.) or vehicle (EtOH; Unsync.) for 18 h. Following synchronization (confirmed by flow cytometry; data not shown), protein was analyzed via Western blotting with antibodies for p-S81, phospho-Ser294 (p-S294), or PR. Right: T47D-YB cells were synchronized as just described (or treated with vehicle; Unsync). Following synchronization, cells were treated for 60 min with vehicle (DMSO) or TBB (10 μM). Protein was analyzed via Western blotting with antibodies for p-Ser81, PR, or Erk1/2 (loading control).