Fig. 4. JC-229 specifically inhibits the ssDNA-binding activity of TbRPA.

EMSA used to measure the ssDNA-binding activity of recombinant RPA proteins (gel images, a, c, e, g) and inhibition of this binding activity by JC-229 (gel images, b, d, f, h) are shown. a ssDNA-binding activity of TbRPA1 DBD-AB. Increasing concentrations (0.05, 0.2, 1.6, 3.2, 6.25, 12.5, 25, 50, and 100 nM) of TbRPA1 DBD-AB protein were incubated with 2 nM oligo dT₃₂ labeled with 5´IRDye (5´IRDye800-dT₃₂) and the protein–ssDNA complex was analyzed by EMSA. b Inhibition of the ssDNA-binding activity of TbRPA1 DBD-AB by JC-229. Serial dilutions (0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 μM) of JC-229 were pre-incubated with 12.5 nM TbRPA1 DBD-AB protein, then incubated with 5´IRDye800-dT₃₂. Inhibition of binding was visualized by EMSA. c–h The same binding assay and JC-229 inhibition assay were performed with purified HsRPA1 DBD-AB protein (c, d), with the TbRPA complex (e, f) and HsRPA complex (g, h). i, j SDS-PAGE showing purified fractions used for EMSA assays. Three independent EMSA experiments were performed for Fig. 4a–h with similar results. Source data are provided as a Source Data file.