Fig. 1
DAF-16/FOXO binds to the transcription factor HLH-30/TFEB. a, b DAF-16 binds to HLH-30, preferentially when it is activated by low IIS. Large-scale anti-GFP immunoprecipitations from whole-worm lysates of animals expressing DAF-16::GFP in either a wild type, daf-2(e1370ts), or daf-18(mg198lf) background. Immunoprecipitated material was analyzed by mass spectrometry (LC-MS/MS). In a, spectral counts from all three purifications were combined and the 21 most abundant proteins are shown, including the bait DAF-16 and several of its established binding partners. The arrow indicates the most abundant co-purifying transcription factor, HLH-30. In b, the spectral counts for each purification were kept separate and are shown for the bait, the 14-3-3 protein FTT-2, as well as HLH-30. c Confirmatory co-immunoprecipitation (co-IP). HLH-30::GFP was immunoprecipitated from whole-worm lysates of the indicated C. elegans strains using GFP-Trap resin. Benzonase (50 U ml−1) was added to eliminate DNA- or RNA-mediated interactions. Inputs (IN) and eluates (IP) of the co-IP were analyzed by SDS-PAGE and western blotting. For the inputs (IN), only fractions were loaded: 5% for the anti-FLAG western blot and 50% for the anti-GFP western blot. IB: antibody used for immunoblot. d In vitro binding assay. Recombinantly expressed His6::myc6::HLH-30 was pulled down using Glutathione-Sepharose resin coated either with recombinant GST or GST::HA4::DAF-16. Samples were analyzed by SDS-PAGE and western blotting. For the input, only 50% of the sample were loaded. e Size-exclusion chromatography, illustrating the size distributions and thus complex incorporation of DAF-16 and HLH-30 under different conditions. C. elegans co-expressing DAF-16::FLAG and HLH-30::GFP were treated in the indicated ways, either by use of daf-2(e1370ts) or by exposure for 6 h to either 32 °C or 6 mM t-BOOH. Animals were then immediately frozen, lysed, and their lysates subjected to size-exclusion chromatography on a Superose 6 column. Elution fractions were analyzed by SDS-PAGE and western blotting. Only higher molecular weight fractions are shown. For a full weight-spectrum of untreated animals see Supplementary Fig. 1b
