Bryostatin 1 induces tyrosine phosphorylation and DNA binding activity of STAT1. (A) Whole cell lysates from CLL cells treated with 10 nM Bryostatin 1 for 0.5 to 3 hours were analyzed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) followed by Western blotting using antibodies specific for the tyrosine phosphorylated form of STAT1 (Tyr-P-STAT1), serinephosphorylated form of STAT1 (Ser-P-STAT1), or total STAT1 (bottom panel). Data from the 3 patients' cells shown are representative of results from 21 patients' cells. (B) DNA binding activity of nuclear extracts from CLL cells was examined by EMSA using a radiolabeled probe containing a GAS sequence. Cells were treated with 10 nM Bryostatin 1 as indicated. One hundred molar excess of unlabeled probe (100 × cold GAS) was used for competition experiments. Anti-STAT1 antibody was added to the binding reaction mix to verify that the protein-DNA complex contained STAT1. A control antibody (anti-STAT6) was used to verify specificity.