Figure 7.

Overexpression of SUP35 rescues H₂O₂ stop codon read-through. (A) Sensitivity to oxidative stress was determined by spotting strains onto SRaf or SGal media containing various concentrations of H₂O₂. Results are shown for wild-type and ski2 mutant strains containing an empty vector or overexpressing SUP35. (B) The levels of termination codon read-through were measured using a dual reporter system in wild-type and ski2 mutant strains containing an empty vector or overexpressing SUP35. Strains were grown to exponential phase in minimal media and treated with 0.5 mM H₂O₂ for 6 h. Read-through was quantified using a plasmid that contains tandem Renilla and firefly luciferase genes separated by a single UGA stop codon. Values shown are means ± SD from three independent determinations. Read-through is expressed as a percentage of Firefly/renilla. (C) Protein was isolated from the wild-type and ski2 mutant expressing the Protein A-stop construct and containing an empty vector or overexpressing SUP35. Western blots were probed with a Protein A antibody and a Pgk1 antibody as a loading control. Blots probed with an anti-Sup35 antibody confirm overexpression of Sup35. Quantification is shown for Protein A concentrations relative to Pgk1. Data are the means of three independent biological repeats and error bars denote SD.