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. 2010 Mar 18;5(3):e9757. doi: 10.1371/journal.pone.0009757

Figure 7. 5′ Phosphatase activity of rNSs with DNA substrates.

Figure 7

Panel A: The 5′ phosphatase activity of rNSs was assayed in the absence of ATP as described in the methods section. Lane 1: 5′ end labeled dsDNA (0.3 nM) alone, lane 2–4: dsDNA treated with 1 µg of rNSs (in triplicate), Lane 5–7: 5′ end labeled ssDNA treated with 1 µg of rNSs (in triplicate), lane 8: ssDNA alone. Panel B: The 5′ phosphatase activity of rNSs was assayed in the presence of ATP (1 mM) as described in the methods section. Lane 1: 5′ end labeled dsDNA treated with 5 units (u) of ATPase enzyme, Lane 2–4: 5′ end labeled dsDNA treated with 1 µg of rNSs (in triplicate), Lane 5–7: 5′ end labeled ssDNA treated with 1 µg of rNSs (in triplicate), Lane 8: 5′ end labeled ssDNA treated with 5 u of ATPase enzyme. Panel C The 5′ phosphatase activity of rNSs was assayed with 5′ end labeled siRNA substrate. Lane 1: siRNA alone, Lane 2–5: siRNA treated with increasing concentrations rNSs (1, 2 and 3 µg), Lane 6: CIP treated siRNA. Panel D Effect of dsRNA concentration: rNSs (0.5 µg) was incubated with increasing concentrations of 5′ labeled dsRNA and the activity was measured as described in the methods section. Lane 1: labelled dsRNA alone, Lane 2–6 rNSs incubated with 0.2, 0.4, 0.6, 0.8 and 1 nM dsRNA respectively.