Bryostatin 1–induced STAT1 activation requires MAPK activation and new protein synthesis. (A) CLL cells were pretreated with pharmacologic inhibitors of MEK1/2, PD98059 (PD) or U0126 (U0), for 1 hour and then treated with 10 nM Bryostatin 1 (Br) for 3 hours. Whole cell lysates were prepared and analyzed by Western blotting using antibodies specific for the tyrosine phosphorylated form of STAT1 (Tyr-P-STAT1) or the phosphorylated forms of the MAP kinases, P-p44 and P-p42 MAPK. Blots were stripped and reprobed for total STAT1 and total p44 and p42 MAPK. The results are representative of 5 patients' cells analyzed. Untx indicates untreated. (B) CLL cells were pretreated with 10 μM cycloheximide for 1 hour and then treated with 10 nM Bryostatin 1 for 3 hours. Whole cell lysates were prepared and analyzed by Western blotting using antibodies specific for Tyr-P-STAT1, and blots were stripped and reprobed for total STAT1. Similar results were found in experiments using 4 additional patients' cells.