Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress
Fig 2
Time course of Klotho-mediated phosphorylation of endogenous 14-3-3ζ (Ser-58) effect on 14-3-3ζ monomer levels in HEK 293 cells.
(A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the Trx/ASK1 complex protection against oxidative stress occurred (Fig 1). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 kDa protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in S3 Table.