Fig. 2: Inhibition of nicking activity of RAGs in presence of Dolutegravir.
From: Biochemical activity of RAGs is impeded by Dolutegravir, an HIV integrase inhibitor
a Silver stained gel showing purified RAG1 and RAG2 from human cells. GST-core RAG1 (~94 kDa) and GST-core RAG2 (~65 kDa) are indicated with arrowheads. b. Western blot showing GST-cRAGs. Anti-RAG1 and anti-RAG2 antibodies were used for western blotting. c Sequence of DNA substrate containing 12RSS used for the study. d, e Effect of Dolutegravir on the nicking activity of RAGs on DNA substrate containing 12 RSS. Increasing concentration of Dolutegravir (0.03, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mM) was used for assessing the effect on RAG mediated cleavage on 12 RSS and the products were resolved on a 15% denaturing PAGE. 0.1 and 0.2 mM Elvitegravir was used for comparison of inhibition (d). Bar graph representing effect of Dolutegravir on RAG-mediated nicking of 12RSS (n = 3). Numbers for each bar corresponds to the lane numbers in the panel d. (P values ** 0.001, *** 0.0002, **** <0.0001). f Sequence and structure of heteroduplex bubble substrate used for the study. g. Effect of Dolutegravir on RAG mediated cleavage on heteroduplex DNA. Impact of Dolutegravir on cleavage by cRAG was tested by incubating increasing concentrations of inhibitor (0.1. 0.2, 0.3, 0.4 and 0.5 mM) followed by resolution on a denaturing PAGE. h Bar graph representing inhibition of RAG cleavage of heteroduplex DNA by Dolutegravir (n = 3). Numbers for each bar corresponds to the lane numbers in panel g.
