Fig 1. The effects of secreted Klotho on rotenone (ROT)-induced activation of p38 MAPK and Trx/ASK1 complex levels in HEK 293 cells.
(A) Plots of time course of ROT-mediated activation of p38-MAPK. Experiments were done at least twice and deviations are shown ± SEM. A representative Western blot is shown beneath plot. (B) Depiction of Trx/ASK1 complex levels in HEK 293 cells after pre-incubation with either control buffer alone or 200 pM Klotho/20 mM NAC for 40 min before adding the 5 μM ROT to induce oxidative stress. ASK1 was immunoprecipitated (IP) with rabbit polyclonal ASK1 antibody and the co-precipitated Trx was revealed by Western blot. A representative Western blot of the samples is shown beneath. *p< 0.05 between ROT and Klotho + ROT. (C) Plots of p38 MAPK activation levels in HEK 293 cells treated with either Klotho or NAC as described in section B, with a representative Western blot. *p< 0.05 between ROT and Klotho + ROT. (D) Total lysate levels of Trx prior to IP. Where phosphorylation was studied, the same blot was stripped with RestoreTM stripping buffer (Thermo Scientific) and re-probed with total p38 MAPK antibody for protein normalization. Digitized values of the WB of are shown in S1 and S2 Tables.