Figure 3. Protein-kinase activity for endogenous substrates are reduced in age-1(mg44) F2 homozygotes.

Day-6 adults were harvested, frozen in liquid nitrogen and ground over dry ice. Kinase activity of cleared, sonicated lysates was assessed by γ-³²P-ATP incorporation per 20 μg protein sample, in 1 min at 30°C. Samples were electrophoresed on gels of 10% acrylamide/SDS. (a) Gel stained with SYPRO Ruby (invitrogen) for total protein. (b) ³²P β-image (Molecular Dynamics Storm) from the gel in a, dried under vacuum. (c) Data summary from 2–3 independent expansions each, of strains N2drm, age-1(hx546), F1 age-1(mg44), F2 age-1(mg44) homozygotes, and daf-16 double mutants with each age-1 allele. (d) Additional controls show that in vitro kinase activity is lower in F2 homozygotes of age-1(mg44), even as day-1 adults, than in F1's at adult days 1 or 6; a second mutation, deleting most of the daf-16 gene, restores age-1(mg44) kinase activity to F1 levels; eggs laid by N2DRM or age-1(mg44) F1 adults are intermediate in kinase activity; and dauer larvae have even higher kinase activity than N2DRM adults. Adapted from [30].