(A–C) Genetic analysis of RQC-mediated ubiquitination in ScIVT. ScIVT reactions were prepared using extracts from strains of the indicated genotype, a lysine-containing truncated mRNA substrate, ubiquitin storage buffer (–) or 100 μM recombinant ubiquitin (+), and either protein storage buffer (–) or the indicated purified proteins (+): Ltn1p at 130 nM, Rqc1p at 70 nM, and Rqc2p at 420 nM final concentration. (D) ScIVT reactions were conducted using rqc2Δ extracts, a lysine-free or lysine-containing truncated mRNA substrate, and 100 μM exogenous ubiquitin. After 0 min (t = 0) or 30 min (t = 30) of translation, all reactions were supplemented with an equal volume of ‘mock ScIVT’ (i.e., without mRNA) containing 1.34 μM purified Rqc2p, 100 μM exogenous ubiquitin, and the indicated inhibitor(s). Indicated time points (‘Time (min)') were analyzed by SDS-PAGE and immunoblotting. ‘Long’ and ‘Short’ refer to exposure times of the blots.