Figure 3.

P-TEFb modulates SEC protein assembly. (A) Cdk9 kinase activity is required for the assembly of SEC Cells overexpressing HA-AFF4 were treated with Flavopiridol and subjected to immuno-precipitation with αHA IgG, followed by western blot analysis with the indicated antibodies to detect endogenous SEC and P-TEFb protein subunits. Western blot for input represent 10% of total lysate. (B) KO of cyclin T1 expression disrupts SEC/AFF4 assembly. Wild-type HEK, or cells that are depleted of cyclin T1 (cyclin T1 KO) were transfected with HA-AFF4 and subjected to immuno-precipitation with αHA IgG, followed by western blot with the indicated antibodies for detecting SEC and P-TEFb protein subunits. Western blot analysis for input represents 10% of total cell lysate. (C) AFF4 is phosphorylated by P-TEFb in cells. Phos-tag analysis of HA-AFF4 purified from control cells or cells treated with Flavopiridol. Cells were lysed 48 hours post transfection and subjected to SDS-PAGE analysis and western blot on Phos-tag acrylamide SDS-PAGE (Wako) as described. (D) AFF4 is phosphorylated by P-TEFb in cells. HA-AFF4 full-length and HA-AFF4–300 were expressed in cells, and 48 hours later were harvested and lysed. Cell lysate was subjected to immuno-precipitation with αHA and precipitated proteins were extensively washed before used as substrates for in-vitro kinase assay in the presence or absence of recombinant P-TEFb (Millipore). Recombinant GST-CTD was purified from bacteria and used as a positive control for P-TEFb phosphorylation. Proteins were analyzed on SDS-PAGE and western blot analysis using the “pIMAGO®-biotin Phosphoprotein Detection Kit” for the detection of protein phosphorylation.