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. 2017 Jul 8;8(51):88259–88275. doi: 10.18632/oncotarget.19111

Figure 1. Secreted Klotho construction and expression.

Figure 1

A., Schema for expression of recombinant Skl. Mouse Skl cDNA was fused with cDNA encoding C-terminal Flag and 6xHis and cloned into pAAV. B., Presence of Skl. Recombinant Skl was expressed in HEK293 and mIMCD cells, and its expression was evaluated by western blotting using a Klotho antibody. C., Flag tag expression. The recombinant Skl expressed in HEK293 cells was probed using Flag antibody to confirm the exogenous expression of Skl. D., Subcellular localization of Skl . The HEK293 cell lysates with overexpression of Skl were separated by ultracentrifugation at 100,000 x g. The distribution of Skl in the membrane fraction (P100) and the cytosolic fraction (S100) were analyzed by western blotting using Klotho antibody. E., Skl immune precipitation. Skl and associated proteins were co-immunoprecipated (co-IP) with Flag or Klotho antibody in 1% NP-40 lysis buffer. Co-IP products were subjected to stain-free gels from NEB.