Research Paper Volume 10, Issue 9 pp 2284—2315

Figure 12. Enhanced Sp1 binding to Prdx6 promoter in cells transduced with Sumoylation–deficient Prdx6 against oxidative stress. (A and B) SRA-hLECs were transduced with TAT-HA-Prdx6WT and its mutant TAT-HA-Prdx6K122/142R mutated at Sumoylation sites recombinant protein followed by different concentrations of H₂O₂ (A) or UVB (B) exposure as indicated. ChIP assay was carried out using ChIP grade anti-Sp1 antibody. The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 promoter bearing GC-box (Sp1 sites). Histogram shows the amplified DNA with real-time PCR analysis; open bars vs gray bars vs black bars. The data represent mean ± SD from two independent experiments (**p<0.05; *p<0.001). (C) The H₂O₂ and/or UVB treatment schedule. (D) In vivo DNA binding assay revealed that transduction of Prdx6 and its mutant at K122/142R linked to TAT reactivated binding activity of Sp1 in aging primary hLECs. Primary hLECs of variable ages were transduced with TAT-HA-Prdx6 WT or its mutant TAT-HA-Prdx6K122/142R. ChIP experimentation was conducted using anti-Sp1 antibody. Immunoprecipitated DNA fragments were purified and processed for qPCR analysis using specific primers. Histograms represent the TAT-HA-Prdx6 WT and its mutant-induced enrichment of Sp1 at GC-box (Sp1 binding sites) in Prdx6 gene promoter. Open vs gray and black bars, and gray vs black bar; **p<0.05, *p < 0.001. Data revealed a significant augmentation of Sp1 binding by TAT-HA-Prdx6K122/142R in all ages of LECs, but younger cells were more responsive.