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. 2015 Dec 11;213(8):1307–1314. doi: 10.1093/infdis/jiv588

Figure 1.

Figure 1.

Complex assembling and human Argonaute 2 (hAgo2) activity. A, In human cells, dicer enzyme processes double-stranded RNA (dsRNA; left). The resulting small interfering RNA (siRNA) is loaded by dicer and transactivation response RNA-binding protein onto hAgo2 (RISC complex) to producing target degradation. hAgo2 and single-stranded RNA (ssRNA) are preassembled in vitro, and the complex is introduced into organisms without RISC enzymes, such as Cryptosporidium, to produce silencing (right). B, Complex assembly was confirmed by the electrophoretic mobility-shift assay assay, using ssRNA labeled with FAM. Lanes 1–3, Samples incubated with hAgo2 (0, 50, 250 ng). Lane 4, Molecular markers. C, ssRNA-hAgo2 complex activity is demonstrated by denaturing gel electrophoresis, using an RNA target (50 nucleotides) labeled with carboxyfluorescein. 1, Cp15 target; 2, Cp15-ssRNA with no target; 3, Cp15-ssRNA with hAgo2 alone; 4, Cp15 target incubated with ssRNA alone; 5, Cp15 target incubated with hAgo2 alone; 6, Cp15 target loaded with 0.01 µM of Cp15-ssRNA; 7, Cp15 target loaded with 0.1 µM of Cp15-ssRNA; 8, Cp15 target loaded with 1 µM of Cp15-ssRNA; 9, unrelated target (60 nucleotides) with 1 µM of Cp15-ssRNA. Abbreviation: mRNA, messenger RNA.