Fig 1: Recruitment of EDF1 to collided ribosomes is ZNF598 independent but RACK1 dependent.(A) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HCT116 WT, ?ZNF598 and ZNF598-overexpression (OE) cells treated with 0 µM emetine (untreated, UT, blue traces) or 1.8 µM emetine (low-dose emetine, EL, orange traces) for 15 min; fraction five isolated for monosomes; fractions 6–10 pooled for polysomes. (B) Schematic of polysome proteomics pipeline to monitor fold change in protein intensity in response to emetine treatment between ?ZNF598 or ZNF598-OE and parental (WT) HCT116 cells. (C) Ratio of relative TMT intensity of individual replicates to mean WT-UT intensity for EDF1 in untreated (blue) or low dose (1.8 µM) emetine treated (orange) samples. (D, E) Volcano plot of ratio of fold change in protein abundance in response to emetine treatment between ?ZNF598 and WT cells (D), or ZNF598-OE and WT cells (E). (F) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HCT116-?ZNF598 cells left untreated (blue) or treated with 1.8 µM emetine for 15 min (orange). TCA precipitated fractions analyzed by immunoblotting with EDF1 antibodies (n = 3). (G) Whole cell extracts from untreated (UT) or low-dose of emetine (EL, 1.8 µM, 15 min) treated cells transfected with non-targeting (SCR) or EDF1 siRNAs were analyzed by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. The ubiquitin-modified proteins are indicated. Long, denotes longer exposure. (n = 3) (H) Quantification of relative TMT abundance (n = 4) of the doubly modified diGly eS10-K138/139 peptide (SAVPPGADKKAEAGAGSATEFQFR) normalized to eS10 protein abundance from polysomes of WT and ?EDF1 cells treated with or without low dose emetine (1.8 µM) for 15 min (also see Figure 2—figure supplement 2F). Error bars denote SD for n = 4. (I) UV absorbance across 10–50% sucrose gradients from lysates of HCT116 cells treated with non-targeting (SCR) or RACK1 siRNAs with 0 µM emetine (blue) or 1.8 µM emetine treatment for 15 min (orange). TCA precipitated fractions were analyzed by immunoblotting with EDF1, RACK1, or RPL10A antibodies (n = 3). Total lysates (inset) analyzed for RACK1 depletion after siRNA treatment using RACK1 antibody. See also Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 2—source data 1 and Figure 2—figure supplement 2—source data 1.Figure 2—source data 1.Related to Figure 2A-E and Figure 2—figure supplement 1A-C; Polysome sucrose gradient fraction (pooled) analysis with or without low-dose emetine treatment (1.8 µM, 15 min).16plex TMT-MS3 analysis of HCT116 WT, ?ZNF598, and ZNF598-OE cells.