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. 2016 Jun 20;44(14):6840–6852. doi: 10.1093/nar/gkw566

Figure 3.

Figure 3.

Translational activity of conventional release factors promotes ubiquitination of 60S·NCs. (A) Induction of high molecular weight (HMW) aggregates of Sup35 in PTET-O7-CDC48 cells by overexpression (OE) of PGAL1-driven Sup35FLAG (full length, FL). Cells were shifted to galactose medium for 28 h to induce Sup35FLAG and Dox was added to cultures for an additional 20 h. Cellular lysates were resolved by electrophoresis on a 10% SDS-acrylamide gel to assess expression of Sup35FLAG (SDS-PAGE, bottom) and on a 1.8% SDS-agarose gel to detect its aggregates (SDD-AGE, top) by western blotting. Expression of Sup35-ΔN or empty vector (V) was used as controls. (Band C) Ribosomal species present in cellular lysates from the same cultures as analyzed in (A) were subjected to sedimentation through 15–45% (B) or 15–42% (C) sucrose gradients. Proteins isolated from individual fractions were separated on 10% SDS-acrylamide gels and probed with anti-ubiquitin, anti-Rpl3 and anti-Rps14 antibodies. Representative blots are shown; see Supplemental Figure S2B for another experimental repeat with samples separated on one gel.