Fig. 2.

Oxidative damage is not responsible for age-related decrease in Klotho protein in vivo. a. Reporter plasmids were oxidized (400 μM H₂O₂) prior to cotransfection with renilla luciferase. Unoxidized promoter plasmids (white bars) were processed similarly to oxidized promoter reporter plasmids (grey bars). Following transfection, firefly luciferase was normalized to renilla luciferase in each well. Data in each experiment were compared to relevant unoxidized promoter expression (mean ± S.E.M; n = 4 independent experiments with three replicates per experiment, Student’s t-test, *p < 0.03, **p < 0.003, ***p < 0.0003). b. HEK 293 cells were incubated for 12 h in medium (white bars) or medium containing 50 μM H₂O₂/20 μM FeCl₂ (grey bars) to induce promoter oxidation. Genomic DNA was isolated and incubated with and without Fpg enzyme. GAPDH, Tau, and KL promoters were amplified by PCR and the number of cycles required to cross threshold quantified. Data were normalized to the untreated control (mean ± S.E.M; n = 4 independent experiments with three replicates per experiment; Student’s t-test, *p ≤ 0.002). c. Genomic DNA isolated from the DLPFC white matter of young (white) and old (grey) rhesus monkeys were incubated with and without fpg enzyme. The number of PCR cycles to cross threshold was quantified. Data were normalized to the young animal average (mean ± S.E.M for n = 4 young and n = 8 old animals; Student’s t-test)