Bortezomib/flavopiridol diminishes constitutive activation of STAT3 and STAT5 in CML cells. (A) K562 cells were incubated with 8 nM bortezomib (Btzmb or Bz) ± 200 nM flavopiridol (FP) for 24 hours, after which nuclear extracts were prepared and subjected to EMSA to monitor STAT3/DNA (left) and STAT5/DNA (right) binding activity as described in “Materials and methods.” For C + C′ (lane 1), 100-fold excess of unlabeled specific oligonucleotides was preincubated for 10 minutes with the nuclear extract of untreated cells prior to addition of ³²P-labeled oligonucleotides. For lane 6, nuclear extracts of untreated cells were incubated with TransCruz gel supershift antibody against STAT3 and STAT5 for 30 minutes prior to addition of ³²P-labeled oligonucleotides. Results are representative of 3 separate experiments. (B) K562 (left) and LAMA84 (right) cells were treated with Btzmb (K562: 8 nM; LAMA: 5 nM) ± FP (K562: 200 nM; LAMA: 150 nM) for 24 hours, after which Western blot analysis was performed to evaluate total level and phosphorylation status of STAT3 and STAT5. Each lane was loaded with 30 μg protein; blots were stripped and reprobed with antibodies to actin to ensure equal loading and transfer. An additional 2 studies yielded equivalent results.