FIG 5.
RBM45 specifically bound to ISE3 under in vitro conditions. (A) ISE3 RNA sequence. The ISE3 RNA shown was used in the binding assay. (B) RNA pulldown assay. Biotinylated ISE3 and complementary ISE3 (ISE3-com) were incubated with recombinant RBM45-His protein and were then pulled down by using streptavidin-conjugated beads. Eluted RNA-bound proteins were analyzed by Western blotting for RBM45. The recombinant RBM45-His protein (Input) and the no RNA-pulldown sample were loaded in lanes 3 and 4 as input and bead only controls, respectively. (C to E) Biolayer interferometry. (C) Comparison of binding affinities of the RBM45 protein (2 μM) for the ISE3 and ISE3-com RNAs, respectively. Maltose-binding protein (MBP-His) was used as a negative protein control. (D) BLI sensorgrams showing association and dissociation of the RBM45 protein with ISE3 at different concentrations over time as indicated. (E) Binding parameters used to calculate KD values (ratios of dissociation and association rate constants). The binding experiments were repeated at least three times for calculating the means and standard deviations (SD), by using various concentrations of the protein.