(A) Pol II C-terminal domain (CTD) Kinase assays with the ELL2, ELL3 and AFF4-containing complexes were performed with GST-Pol II C-terminal domain fusion protein (GST-CTD). ELL2, ELL3, or AFF4 complexes were assayed in the presence of ATP and/or the GST-CTD and subjected to Western blot analyses with antibodies specific to Pol II CTD phosphoserine 2 (pS2) and phosphoserine 5 (pS5), CDK9 and AFF4. Consistent with previous observations, serine 2 and serine 5 of Pol II CTD are good substrates for the P-TEFb complexes in vitro. CDK9, itself, is also known to be autophosphorylated, resulting in a shift in gel migration in SDS- PAGE (indicated by asterisk, while an arrow indicates the faster migrating unphosphorylated form). AFF4 shows a similar gel mobility shift as CDK9, also indicated by an asterisk, suggesting that it is a substrate for P-TEFb as well. See for additional kinase assays. (B) Sequence alignment of a potential site for multiple phosphorylation of AFF4-related proteins bearing SP motifs favored by P-TEFb. (C–D) AFF4, and not AFF1, is required for stability of the SEC containing ELL1, P-TEFb and MLL-partners in HeLa cells. Western blot analysis of ELL1, CDK9 and Cyclin T1 was performed in the presence and absence of AFF1 or AFF4. Nuclear extracts from the siRNA-mediated for AFF4 or AFF1 were analyzed by SDS-PAGE and Western blot analysis. Arrows indicate increasing protein loads. Bulk protein levels of ELL1 are reduced in AFF4, but not AFF1 knockdown in these cells. Bulk protein levels of P-TEFb were not affected by AFF1 or AFF4 RNAi. Global H3K4 and H3K79 methylation levels were not affected by AFF4 knockdown. Tubulin serves as a loading control. (E) Gel filtration analyses of nuclear extracts from control and AFF4-directed siRNA treated cells. Larger P-TEFb-containing complexes, SEC (fractions 10–14 also seen in and indicated by underlining in red) are reduced in AFF4 knockdown cells, indicating that the presence of AFF4 is required for the assembly of SEC.