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Pre-treatment of HGF with trisulfides and nitration Recombinant mouse HGF (carrier protein-free) was pre-treated with glutathione trisulfide (GSSSG) and lipoic acid trisulfide (LASSS) at mole ratios (HGF : trisulfide) of 1:0 (with solvent alone), 1:400, 1:1000, and 1:4000 at pH 7.4, 25C just prior to the peroxynitrite protocol (optimized at a molar ratio 1:2000 relative to HGF, pH 7.4, 37, for 30 min), which was previously demonstrated to induce maximal nitration of Y198 and Y250 located in the -chain under physiological conditions as described previously 32
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this, in turn, resulted in the upregulated expression of telomere repeat-binding factor 1/2 (TRF1/TRF2) proteins and strict dependence on p53/p21 signaling, followed by irreversible G2/M cell cycle arrest, ultimately leading to apoptosis
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