(A) [35S]-methionine incorporation into early-log cells grown for 2 hr (∼3 doublings) in M9 media at 37°C. Immediately prior to the radioactivity pulse, cultures were treated with increasing concentrations of lamotrigine. [35S]-methionine incorporation was quantified by liquid scintillation counting. Error bars represent the error of two biological replicates. (B) Cell-free coupled transcription/translation reactions in the presence of increasing concentrations of lamotrigine. Samples were incubated at 37°C for 1 hr, at which time reactions were halted on ice, excess luciferin was added, and luminescence was monitored. Error bars represent the error of two biological replicates. (C) Kinetics of cell-free transcription/translation system at 15°C. 10 μl reactions containing 1% DMSO were read every 30 min for 5 hr to establish a linear range of luciferase production. Error bars represent the error of two biological replicates. (D) Example, pulse-labeling data showing the incorporation of 50% 15N into an r-protein peptide as a function of time. Similar data were gathered for all r-protein peptides from cells treated with DMSO; 2×, 3×, 4×, 5×, and 6× MIC lamotrigine; and 2×, 3×, 4×, 5×, and 6× MIC chloramphenicol. (E) R-protein degradation as a function of time in DMSO-treated cultures. Protein degradation is defined as (14N intensity/15N intensity) for each peptide. Light shades to dark shades represent 0, 4, 8, and 16 hr of 50% 15N pulse. Small circles represent individual peptide measurements. Large circles denote the median measurement for that sample. (F) R-protein synthesis as a function of time in DMSO-treated cultures. Protein synthesis is defined as (50% 15N intensity/15N intensity) for each peptide. Light shades to dark shades represent 0, 4, 8, and 16 hr of 50% 15N pulse. (G and I) are the same as (E), except for 6× MIC lamotrigine and 6× MIC chloramphenicol, respectively. (H and J) are the same as (F), except for 6× MIC lamotrigine and 6× MIC chloramphenicol, respectively.
DOI: http://dx.doi.org/10.7554/eLife.03574.020