Figure 3. The P-TEFb binding region of Brd4 causes a release of P-TEFb from the 7SK snRNP in vivo.

HeLa cells were transfected with plasmids expressing Brd4 1209–1362 (Brd4) or mutant Brd4 1209–1362 Δ1329–1345 (Brd4Δ). A) Immunofluorescence microscopy. 48 hours after transfection cells were fixed and stained for DNA (DAPI) or the FLAG tagged Brd4 constructs (FLAG). B) Glycerol gradient sedimentation analysis. Cell lysates were prepared 48 hr after transfection and sedimented on 5–45% glycerol gradients as described in Materials and Methods. Western blots of fractions were probed with antibodies to LARP7 or Cdk9 as indicated.