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. 2018 Nov 1;72(3):469–481.e7. doi: 10.1016/j.molcel.2018.08.037

Figure 5.

Figure 5

ZNF598 Detects Collided Ribosomes Induced by Multiple Types of Stalls In Vivo

(A) Strategy to induce complete versus stochastic ribosome stalling in cells treated with translation elongation inhibitors.

(B) Wild-type (WT) or ZNF598-knockout (ΔZNF598) HEK293 cells were pre-treated for 15 min with low (1.8 μM) or high (360 μM) emetine, lysed, digested with micrococcal nuclease, and separated by sucrose gradient centrifugation. The 260 nm absorbance profiles across the gradient are normalized to the 80S mono-ribosome peak.

(C) HEK293 cells pre-treated for 15 min with low or high concentrations of the indicated elongation inhibitors were analyzed by immunoblotting. Ubiquitinated eS10 (Ub-eS10) was detected using more sensitive reagents than unmodified eS10. For reference, ∼10% of total eS10 is ubiquitinated in low-dose inhibitor-treated samples and ∼1%–2% in untreated samples.

(D) Cytosol from HEK293 cells pre-treated for 15 min with nothing (top), high (middle), or low (bottom) concentrations of emetine were separated by sucrose gradient fractionation and immunoblotted for ZNF598 and uL2.

See also Figure S6.