Fig. 6.

Nanobodies inhibited TUT4 uridylation activities in vitro. (A) In vitro TUTase assay with a titration of Nb-S2A4 at 13.3 nM, 33.3 nM, 66.7 nM, 133 nM, 200 nM, 333 nM, 733 nM, 1.33 µM, and 2 µM. (B and C) In vitro TUTase assay with addition of gradient concentrations of both the nanobody inhibitor and complex binders at 3.3 µM, 8.3 µM, and 20 µM. Nb.BV025 and Nb-R1A12 were used as negative controls. (D) In vitro pan-uridylation assay of pre-let-7g carried out by full-length TUT4, 1.6 µM LIN28 with unlabeled pre-let-7g. (E) In vitro pan-uridylation assay of mRNA SHOC2-A10 carried out by full-length TUT4, 1.6 µM LIN28, and unlabeled mRNA SHOC2-A10.