Journal: Nucleic Acids Research
Article Title: eIF2D promotes 40S ribosomal subunit recycling during intrinsic ribosome destabilization
doi: 10.1093/nar/gkaf1322
Figure Lengend Snippet: Decreased IRD-inducing protein levels and altered splicing patterns in eIF2D-deficient cells. ( A ) Cumulative fraction of the fold changes in protein levels in eIF2D KO cells relative to the control cells ( n = 4 biologically independent samples). Protein levels were analyzed via DIA-MS, and the results are shown for all detected, short nascent chain, and long nascent chain proteins. P -values were calculated using the two-tailed Mann–Whitney U test. ( B ) Differences in the median log 2 fold changes in protein levels between the indicated cell lines and control cells. ** P < .01 and *** P < .001; NS, not significant (two-tailed Mann–Whitney U test). ( C ) Gene Ontology (GO) term analysis of IRD target genes with reduced protein levels in eIF2D KO cells. FDR, false discovery rate. ( D ) Volcano plots showing the differences in the PSI scores of skipped exons between the eIF2D KO and control cells ( n = 2 biologically independent samples). Gray dashed lines indicate a ∆PSI of ±0.05 and an FDR of 0.05. ( E ) Numbers of skipped and retained introns in the eIF2D KO, eIF2A KO, and MCTS1 KO cells compared to those isplicing, and transcripn the control cells. Alternative splicing events not consistently altered in two independent clones were excluded. ( F ) Cleavage and polyadenylation specific factor 2 ( CPSF2 ) gene structure and Sashimi plots showing the differential inclusion of CPSF2 exon 14. Representative data of two replicates are shown. ( G ) Immunoblotting analysis of WT, eIF2D KO, and MCTS1 KO cells using the indicated antibodies. HSP90 was used a loading control.
Article Snippet: Primary antibodies against MCTS1 (#GTX117793; GeneTex), DENR (203057-T46; Sino biologicals), eIF2D (12840-1-AP; Proteintech), CPSF2 (A301-581A; Bethyl Laboratories), HSP90 (610 419; BD Biosciences), and V5 epitope (R960CUS; Thermo Fisher Scientific) were used in this study.
Techniques: Control, Two Tailed Test, MANN-WHITNEY, Alternative Splicing, Clone Assay, Western Blot