Figure 4.

AFF4-CHD is a substrate of P-TEFb kinase. A, time course of the phosphorylation of AFF4-CHD by P-TEFb. AFF4-CHD and SPT4/SPT5 were incubated with P-TEFb over a time course of 2 h, and samples were taken at 0, 30, 60, and 120 min. SPT5 serves as a control to monitor the activity of P-TEFb and demonstrate the shift of protein bands after incubation with P-TEFb in the presence of ATP. B, final products of the phosphorylation assay triplicates for the identification of phosphorylation sites using MS. Lanes 1 and 5 are apo proteins before adding P-TEFb. Lanes 2–4 and 6–8 are the triplicates of the phosphorylation assay after 2 h of incubation. C, binding of AFF4-CHD to 10 nm fluorescence labeled FBS 35-mer G-quadruplex in apo and phosphorylated states. The binding assay was performed after incubating AFF4-CHD with WT P-TEFb kinase or catalytical mutant of P-TEFb kinase in the presence of ATP. The experiments were repeated three times, each time with two pipetting repeats as in Fig. 3C. D, the final products of the three phosphorylation triplicates of the AFF4-CHD incubated with P-TEFb and P-TEFb–N-mut were injected onto the gel filtration column (Superdex 200 increase 3.2/300; GE Healthcare), respectively.