daf-16d/f is regulated at the level of transcription in both a temporal and spatial manner. (A,B) Quantitative RT-PCR analysis of endogenous daf-16a and daf-16d/f mRNA levels during larval development (L1-L4) and in the adult. In both wild-type and daf-2(e1370) mutants, the level of daf-16d/f mRNA significantly increases with age. Additional details are shown in Additional file 1: Figure S7. (C,D) Changes in DAF-16d/f and DAF-16a protein levels with age. Equal volumes of worms were used for each larval stage. Western blots with the worm lysates were probed with anti-α-DAF-16 antibody (top panel) and anti-α-tubulin (bottom panel). DAF-16d/f shows a dramatic increase in protein levels with age as compared to DAF-16a. (E) Quantification of the fluorescence intensity in Pdaf-16a::gfp and Pdaf-16d/f::gfp transgenic worms as they age The GFP intensity was quantified using ImageJ software and normalized to the L1 stage for each strain. (F) Transcriptional regulation of daf-16d/f determines longevity. daf-16(mgDf50); daf-2(e1370); daf-16d/f transgenic worms were treated with diluted daf-16 RNAi bacteria from the L4 stage to day 2 adulthood. All lifespan data are shown in Additional file 1: Table S5.