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. Author manuscript; available in PMC: 2018 Jun 1.
Published in final edited form as: Cell. 2017 Jun 1;169(6):1051–1065.e18. doi: 10.1016/j.cell.2017.05.022

Figure 1.

Figure 1

Affinity enrichment of mammalian ribosomes defines the ribo-interactome in ESCs.

A, In mouse ESCs, eL36 and eS17 are endogenously tagged with FLAG using CRISPR-Cas9 endonuclease system denoted by scissors. In addition to the endogenously FLAG tagged RPs, cells stably expressing different levels of GFP-FLAG transgenes were generated using PiggyBac transposon-mediated stable integration. GFP-FLAG transgene clone 3, expressing FLAG at similar levels to the tagged RPs, was chosen for further analyses.

B, Strategy to define the mammalian ribo-interactome. GFP-FLAG cells are used to assess the background of the ribosome affinity enrichment strategy. Cytoplasmic lysates from eL36-FLAG, eS17-FLAG, and GFP-FLAG cells are subjected to FLAG IP under similar conditions, and IPs are analyzed by LC/MS-MS. Average, normalized spectral abundance factor (NSAF) of RPs from three biological replicates of either eL36-FLAG or eS17-FLAG are shown. See Table S1.

C, Maximum SAINT probability scores and fold enrichment of eL36 and eS17 experiments are shown. SAINT probability of 0.56 corresponds to 0.08 FDR. 60S RPs are colored in blue, 40S RPs in yellow.

D, eL36 specific interactors are defined as those present in all eL36 biological replicates with at least 2 unique peptides, but not present in any of the eS17 biological replicates. The overlap between eL36 and eS17 datasets is defined as the proteins present at the intersection of at least one eL36 and one eS17 replicate with a SAINT score >=0.56. For GO biological process analysis, Benjamini–Hochberg FDR cutoff of 8% and fold enrichment >=4 are used. Examples of enriched GO categories are shown, for a full list see Table S2. The number of identified genes in each GO category is shown in comparison to the number of genes in each GO category.