Figure 6
From: Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin
STI treatment lowers transfection and transformation efficiency of plasmid DNA. Following incubation without (−) or with STI (S1 or S2) for 0 or 2 h, the plasmid pGL3-PKD1 (pGL3 inserted with a 200 bp PKD1 proximal promoter to drive expression of Luciferase) DNAs were purified by a PCR reaction purification kit and analyzed by agarose gel (A). The purified DNA (0.1 ug) was used in co-transfection with β-galactosidase (0.1 ug) cDNA in HEK293T cells. The expressed luciferase activity in each was assayed and normalized with co-expressed β-galactosidase activity. The normalized RLU value obtained in cells transfected with untreated pGL3-PKD1 was set at 100% (B). Each bar is the mean ± S.D (**p < 0.005) of a representative experiment performed in triplicate. (C) 50 ng the purified STI-treated DNA was used for transformation of XL1-blue competent cells. Each symbol with matching color in both 0 h and 2 h represents an independent experiment. The bar is the mean of the data in each41 (Kueh et al. Science 341:670-673, 2013). (D) DNA nicking activity by bovine Aprotinin (Ap) and recombinant Aprotinin (rAp) overexpressed in Nicotania. (E) Transfection efficiency of the pGL3-PKD1 treated with Ap or rAp. The expressed luciferase activity was assayed and normalized as in B (**p < 0.005). Samples in each gel were prepared in parallel. All gels were processed using Microsoft Word “Corrections” for brightness and contrast applied equally across the entire image including controls. The full length gels are presented as Supplementary Information with corresponding figure numbers as in the main article.
