Figure 6.

JBP2 stimulates HOMedU formation in vivo. (A) Specificity of the HOMedU antibody was evaluated by immunoprecipitation assays using ³²P-labeled mononucleotides derived from oligos containing HOMedU (HMU), base J (J) or thymidine (T) (see Methods section for oligo sequences). Postlabeled mononucleotides were incubated with α-HOMedU antibody conjugated to paramagnetic beads, washed to remove nonspecifically bound nucleotides followed by scintillation counting. Data are representative of three independent assays. Data are background subtracted and adjusted to results from unmodified DNA (T). (B) Anti-HOMedU immunoprecipitation analysis was used to examine HOMedU content of WT and the indicated mutant T. brucei cell lines. Genomic DNA was isolated from the indicated cell lines and analyzed for HOMedU content as in panel A. J-null (Null); J-null expressing WT JBP2GFP (Null+JBP2); J-null feed HOMedU (Null+HOMedU); J-null expressing JBP2 H441A mutant (Null+ JBP2 TH mutant). Each IP was repeated in triplicate and average ± SEM is shown.