Figure 4.

P-TEFb modulates AFF1/AFF4 dimerization. (A) AFF1–AFF4 protein dimerization HA-AFF4 and Flag-AFF1 were co-expressed in HEK293T. 48 hours post transfection, cells were lysed and subjected to immuno-precipitation with αHA IgG. IP reactions were analyzed by western blot with the indicated IgG to confirm protein association. Input represents 10% of total cell lysate. Both immuno-precipitated and input samples were also analyzed with AFF1 or AFF4 IgG, to confirm that proteins detected on the gel are indeed AFF1/4. (B) P-TEFb modulates AFF1/AFF4 protein dimerization. Cyclin T1 KO HEK cells were transfected with either HA-AFF4, Flag-AFF1, or both proteins. Cell lysate was subjected to αFlag IP followed by western blot with αHA IgG. (C) Kinase active Cdk9 is not required for AFF1 /AFF4 protein dimerization. Flavopiridol-treated cells (50 mM) were co-transfected with Flag-AFF1 and HA-AFF4, and subjected to IP with αFlag IgG. Immuno-precipitated samples were analyzed by western blot with αHA to confirm the association between AFF1 and AFF4. Input samples represent 10% of total cell lysate and were also analyzed by western blot with both αFlag and αHA IgG. (D) Domain mapping of AFF4 regions that mediate dimerization with AFF1. Flag-AFF1 was co-expressed with the indicated HA-AFF4 - C- terminal truncated mutants and subjected to IP with αFlag IgG. IP samples were analyzed with αHA to confirm association of AFF1 and AFF4 mutant proteins. Input represents 10% of total cell lysate and was also analyzed by WB with both αFlag and αHA IgG.