Fig. 4.
Competition of p19 with human siRNA-containing complexes found in cytosolic HeLa cell extract. (a) Normalized fraction of siRNA bound to complexes D (green), C2 (red), and C1 (black), where the highest point is normalized to unity in each complex over time, as derived from the gel in panel B. The relative abundances of C1 and complex D increase over time with estimated rate constants of 8 × 10−4 s−1 and 4 × 10−4 s−1, respectively, whereas C2 decreases in relative abundance with a rate constant of 2 × 10−3 s−1. (b) Formation of complexes D, C2, and C1 after 10, 30, 60 and 120 min incubation of siRNA in cytosolic HeLa cell extract. Samples were loaded onto a running 4% native polyacrylamide gel, leading to the indicated differences in migration. (c) Anti-Dicer antibody-based supershift of siRNA-containing complexes. Dicer containing complex D shifts in the presence of anti-Dicer antibody (lane 3: 0.8 μg; lane 4: 1.6 μg). Lanes 1, 5 and 6 indicate the mobility of the complexes in the absence of any antibody. Lanes 9–12 show negative controls where 0.8 μg or 1.6 μg of anti-actin (lanes 9, 10) or anti-DsRed (lanes 11, 12) were added. (d) Quantification of the normalized fraction of siRNA bound in each complex, as derived from gels as in panel E. Fractions were normalized to the zero time point. (e) Formation of complexes after 2.5 h incubation of siRNA in cytosolic HeLa cell extract, and increasing concentrations of p19. (f) Quantification of the normalized fraction of siRNA bound in each complex, as derived from gels as in panel E, normalized as in D. (g) Formation of complexes D, C1, and C2 after 2 h incubation of siRNA in cytosolic HeLa cell extract, followed by the addition of increasing concentrations of p19 and further incubation for 30 min.