Bortezomib/flavopiridol inhibits phosphorylation of the C-terminal domain (CTD) of RNA polymerase II, an action mimicked by the P-TEFb inhibitor DRB. (A) K562 (top) and LAMA84 (bottom) cells were incubated in the presence of flavopiridol (FP, K562: 200 nM; LAMA: 150 nM) ± bortezomib (Btzmb or Bz, K562: 8 nM; LAMA: 5 nM) for 24 hours, after which Western blot analysis was performed to monitor phosphorylation of CTD. (B) K562 cells were exposed to 8 nM bortezomib (Btzmb) ± 30 μM DRB for 48 hours, after which percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright Giemsa-stained cytospin preparations. Results represent the means ± SDs for 3 separate experiments performed in triplicate. (C) Alternatively, K562 cells were treated as described for panel B, and apoptosis was assessed by Annexin V-FITC staining and flow cytometry as described for Figure 1A. Results are representative of 3 separate experiments. (D) K562 cells were treated as described for panel B, after which cells were lysed and subjected to Western blot using the indicated primary antibodies. CF indicates cleavage fragment. For panels A and D, 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.