Effect of phosphorylation site mutation on the interaction of Ac -FTT-2 with Ac -DAF-16. HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels, Western blot with anti-human 14-3-3β antibody; Bottom panels, the blot was stripped and re-probed with Ac-DAF-16 antiserum. See text for description of the Ac-DAF-16 mutants.