FIGURE 1

Treatment of GTP in vitro to human skin fibroblasts HS68 cells inhibits UVB and H₂O₂ induced protein oxidation. Immunoblot analysis of carbonyl residues was performed in: Panel A, protein extracts from UVB irradiated (30 mJ per cm²), Panel B, H₂O₂ treated (25 M), and Panel C, H₂O₂ (25 M) and catalase (100–400 U per mL) treated human fibroblasts. HS68 cells were treated with GTP for 24 h thereafter washed with PBS to remove GTP. Cells in PBS buffer were irradiated with UV. After UV irradiation, cells were again treated and cultured with GTP for 24 h, and thereafter washed with PBS and harvested for the analysis of protein oxidation. In case of H₂O₂ treatment, first cells were treated and cultured with GTP for 24 h thereafter washed with PBS. After washing, cells were treated with H₂O₂ for 12 h without GTP. After 12 h of H₂O₂ treatment, cells were washed and cultured again with GTP for next 24 h in medium. Again cells were washed with PBS and harvested for western blot analysis of protein oxidation. Five g of protein extracts were incubated with DNPH, subsequently electrophoresed by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with polyclonal rabbit anti-dinitrophenylhydrazone antibody, as detailed in Material and Methods. A representative blot from three independent experiments with identical results is shown. Treatments are as described in the figures.