1. Functional assays confirm daf‐2‐dependent reduction in protein metabolism.
Functional assays confirm daf‐2‐dependent reduction in protein metabolism. (A) Representative traces of polyribosome profiles obtained from N2 (black), daf‐2(e1370) (blue), and daf‐16(mu86); daf‐2(e1370) (red) nematodes harvested at day 1 of adulthood (identical conditions as described for the proteomics experiments). (B) Quantification of the peak (P) heights in the obtained polyribosome profiles using the starting point before the 40S peak (marked by an arrowhead, Figure 2A) as the zero value in at least four independent experiments. Values represent mean±s.e.m. All measured peaks are significantly lower in daf‐2(e1370) compared with N2 (P<0.0002) and this response is dependent on daf‐16 (P< ) as tested by Student's t‐test. a.u., arbitrary units. (C) The daf‐2(e1370) mutant contained significantly less mRNA than N2. mRNA yield (ng/μl) was determined from 1500 nematodes of each strain in triplicate, using poly(A)+ trapping. (D) Total protein levels are similar in all three strains. Protein extracts from 1500 synchronized young adults were determined by spectrophotometric analysis. (E) The daf‐2(e1370) has lower 20S proteasomal core complex activity. 20S proteasomal activity was measured in triplicate on 1500 synchronized day 1 adults for each strain using fluorescence analysis. Statistical significant differences represent means±s.e.m. as calculated with Student's t‐test (*P<0.05).
Gerdine J Stout et al. Mol Syst Biol 2013;9:679
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