Extended Data Fig. 4 ∣. Translation monitoring in a UBAP2L knockout replicate line, replicate concordance, and validation of polysome analyses in UBAP2L knockouts.
a, Translation monitoring using puromycin incorporation. Anti-puromycin western blot of extracts from puromycin-treated UBAP2L knockout (KO1) and parental (WT) HEK293T cell lines. GAPDH served as loading control. b,c, Polysome profile of UBAP2L after treatment of cells with 0.5 mM puromycin (b) and treatment of lysates with 30 mM EDTA (c). Top, absorbance (at 260 nm) plot of a HEK293T cell lysate fractionated through a 10-50% sucrose gradient. Bottom, western blots of UBAP2L from the corresponding fractions. d, Polysome profiles of HEK293T cells (WT, n = 2) and UBAP2L knockout HEK293T cells (KO, n = 4) fractionated through 10-50% sucrose gradients. Light-colored lines indicate means from each set (WT, light blue; KO, pink), and darkly shaded areas denote s.d. (WT, blue; KO, red). e, Bar graphs showing percentages of transcripts with RPKM ≥ 1 of all transcripts with ≥10 reads per transcript for two UBAP2L knockout lines (KO, 2 replicates each) and control samples (WT, two replicates). f, Scatter plots showing correlation of log2-transformed ratios of input-normalized polysome transcript levels (RPKM) between the two UBAP2L knockout HEK293T lines. R, Pearson correlation coefficient. g, Bar graph showing the percentage of regulated transcripts in UBAP2L targets and nontargets. *P < 0.0001 (χ2 test with Yates’s correction). h, RT–qPCR validation of reduced polysome association for the indicated transcripts. Transcript levels in inputs and polysome fractions were measured for KO and WT samples. KO/WT ratios of input-normalized polysome association of transcripts were then calculated. i, Western blots of DDX54, EIF4G1, EIF3B, and EEF2 in UBAP2L knockout cells (KO1, KO2). GAPDH served as a loading control. j,k, Quantitative flow cytometry reporter assay for mRNA translation using RCas9-fused 4EBP1. j, Plasmid design for the RCas9-4EBP1 experiment. k, Bar graph showing mean YFP levels in RCas9-4EBP1-expressing cells, normalized to RCas9-expressing cells, on each targeting site. Error bars denote s.d. from n > 5,000 RCas9-4EBP1-expressing and n > 5,000 rCas9-expressing cells per site. *P < 0.005; n.s., not significant (P > 0.5); two-tailed Student’s t-test. Uncropped images for a–c and i and data for graphs in h and k are available as source data online.