Fig. 3.
In vitro assays for inhibition of trans-translation and translation. (A) DHFR genes with or without a stop codon were expressed in vitro in the absence or presence of additional tmRNA and SmpB. The locations of DHFR, as determined in control reactions, and of the lower-mobility tagged DHFR protein are indicated. (B) In vitro trans-translation reactions were performed after addition of 1.67% DMSO or 10 μM compound. Representative reactions are shown. The intensity of the DHFR and tagged DHFR bands were measured, and the tagging efficiency was calculated as the percentage of total DHFR protein in the tagged DHFR band. The average tagging efficiency with SD for at least three repeats is shown. To determine the dose–response behavior, in vitro trans-translation reactions performed after addition of KKL-35 at different concentrations, and the tagging efficiencies were calculated. (C) A representative experiment is shown. (D) Data from three repeats were averaged, graphed, and fit with a sigmoidal function to determine the IC50. Whiskers indicate SD for each point. (E) In vitro translation reactions were performed after addition of DMSO, 100 μM chloramphenicol (chlor), or 100 μM compound, and a representative experiment is shown. The amount of DHFR in each lane was measured as a percentage of the amount in the DMSO-treated control, and the average value for three experiments was graphed with whiskers indicating the SD. (F) Inhibitor-treated reactions did not result in significantly different translation activity relative to the DMSO-treated control; P values from one-way ANOVA tests are indicated.