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. 2023 Jul 20;186(15):3227–3244.e20. doi: 10.1016/j.cell.2023.05.035

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© 2023 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure S3 — Effects of 3′ UTR translation on mRNA and protein levels, related to Figure 3

(A) mRNA-seq analysis of C. elegans reporter strains expressing YFP-UTR and YFP-STOP (n = 2). Error bars represent mean ± SEM.

(B) qPCR analysis of copy number of YFP-UTR and YFP-STOP in C. elegans reporter strains. Integrated gene copy numbers are on average 1.96 ± 0.11-fold higher in YFP-UTR compared with YFP-STOP. Error bars represent mean ± SEM (n = 3). Data were analyzed using the 2^(−ΔΔCt) formula. p values by unpaired t test.

(C) qPCR analysis of YFP-STOP and YFP-UTR mRNA levels in wild-type C. elegans and in skih-2 mutant animals. Error bars represent mean ± SEM (n = 3). Data were analyzed using the 2^(−ΔΔCt) formula. p values by Fisher’s LSD test.

(D) Representative immunoblot analysis of wild-type or gcn-1(nc40) mutant worms expressing YFP-UTR. See Figure 3C for quantification.

(E) Representative trace of sucrose density gradient fractionation (A_254 nm; top) and immunoblot analysis of 3x-FLAG-tagged GCN-1 in C. elegans. Dotted lines delineate polysome fractions.

(F) Representative traces of sucrose gradient density fractionation of wild-type and gcn-1(nc40) mutant animals (A_254 nm). Dotted line indicates polysomes collected for subsequent MS/MS analysis shown in Figures 3G and 5E.