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. 2020 Dec 8;40(1):e105179. doi: 10.15252/embj.2020105179

Figure EV1. Enrichment of ABCE1 and eIF3j on 40S complexes and assessment of their role in splitting of 80S ribosomes.

Figure EV1

  1. Total cellular extracts from yeast cells expressing ABCE1‐TAP were separated on a sucrose gradient (10–50%) by ultracentrifugation. Proteins of each fraction were analyzed by Western blot using a PAP antibody for the detection of the ABCE1‐TAP fusion protein and anti‐Nog1 antibody to mark the 60S fraction. AU–absorption units at 260 nm.
  2. Silver‐stained NuPAGE gel showing elution from affinity purification using ABCE1‐TAP performed in Tris or HEPES buffer (see Methods for details).
  3. Volcano plot showing the fold enrichment of proteins in the elution fraction from the ABCE1‐TAP purification in Tris buffer followed by mass spectrometry analysis (LC‐MS/MS). The enrichment was calculated relative to an “input” corresponding to an aliquot of the ABCE1‐TAP cell lysate used for the affinity purification. It is represented, on the x‐axis, as log2(LFQ ABCE1‐TAP/LFQ input) where LFQ stands for label‐free quantification. The y‐axis represents the P‐value distribution (‐log10 ‐p‐value) calculated using Student’s t‐test for all identified proteins represented by a circle. Proteins above the curved lines show a statistically significant enrichment according to the t‐test value. The assay was performed in triplicates.
  4. UV profiles from in vitro splitting reaction triplicates with and without splitting factors (SF; ABCE1, Dom34, Hbs1, and eIF6) and eIF3j. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation. (+) = 4‐fold molar excess of eIF3j; (++) = 20‐fold molar excess of eIF3j.
  5. Relative abundance of 80S and subunits in each experiment, as calculated from triplicates shown in (D).
  6. SDS–PAGE of the 40S, 60S, and 80S peaks obtained from the in vitro splitting experiment (D) containing SFs and high amounts of eIF3j.
  7. UV profiles from in vitro “facilitated” splitting reactions. Samples were separated on a sucrose gradient (10–50%) by ultracentrifugation.
  8. SDS–PAGE of the input factors (eIF3j and ABCE1) as well as 40S and 80S peaks from the “facilitated” splitting experiment. ABCE1 and eIF3j are marked by arrows, respectively.