Journal: Science advances

Article Title: Hsc70-4: An unanticipated mediator of dsRNA internalization in Drosophila .

doi: 10.1126/sciadv.adv1286

Figure Lengend Snippet: Fig. 4. Effect of nucleotide substrates on rHsc70-4 binding to dsRNA and JG-98 treatment on silencing of luciferase. EMSAs testing the binding of rHsc70-4 (2 μM) to dsRNA-Cy3 (dsCat, 0.76 nM) in the presence of (A) ATP-γ-S, (B) AMP-PNP, or (C) ADP. Concentrations of 1, 2.5, 5, and 10 mM of each substrate were used. The electropho- retic shift of dsRNA-Cy3 was assessed by native PAGE. All EMSA experiments were performed at least three times, giving similar results. The first lane in all gels corresponds to dsRNA-Cy3 incubated without rHsc70-4. The last lane in all gels corresponds to dsRNA-Cy3 incubated without rHsc70-4 and with each substrate (10 mM). (D) Effect of pretreatment of S2Xpress cells with a JG-98 inhibitor on silencing of luciferase. Experiments were performed as in Fig. 1C. After transfection with luciferase-expressing plasmids, cells were incubated with JG-98 (0.125, 0.25, 0.5, and 1 μM) or DMSO (1 μM). Then, dsFluc or dsCtl was added to the media. Last, luciferase activity was measured. As positive and negative controls of RNAi silencing, dsFluc and dsCtl were cotransfected, respectively. The histogram shows the mean + SD firefly/Renilla ratio relative to Soak. dsCtl from three independent experiments (n = 9). ANOVA followed by Tukey’s post hoc test was used to detect significant differences between treatments. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: For nucleotide substrate assays, the labeled probe (0.76 nM) was incubated with rHsc70- 4 protein (2 μM) and increasing concentrations of ATP- γ- S (11162306001, Sigma- Aldrich), ADP (A2754, Merck), or AMP- PNP (10102547001, Merck) in PBS.

Techniques: Binding Assay, Luciferase, Clear Native PAGE, Incubation, Transfection, Expressing, Activity Assay