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. 2022 Oct 7;298(11):102575. doi: 10.1016/j.jbc.2022.102575

Figure 4.

Figure 4

AcrIF4 prevents the recruitment of Cas2/3.A and B, EMSA to test the Cas2/3 recruitment by the Csy-dsDNA (or Bubble dsDNA) complex or in the presence of AcrIF4. Reactions were performed with 1.6 μM Csy complex, 0.1 μM 54-bp dsDNA (5′-FAM in the target DNA strand), 25.6 μM AcrIF4, and concentrations of Cas2/3 were set as 0.2, 0.4, and 0.8 μM, following the order indicated by the black triangle. Reactions were repeated independently three times with similar results. C, the order of addition of AcrIF4 affects the inhibitory effect confirmed by an EMSA assay. Reaction was performed with 1.6 μM Csy complex, 0.8 μM Cas2/3, and 0.1 μM 54-bp dsDNA bubble (5′-FAM in the target DNA strand). AcrIF4 was added at the indicated order and with concentrations of 0.8, 3.2, and 12.8 μM. The experiments were repeated independently three times with similar results. EMSA, electrophoretic mobility shift assay.