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. 2012 Jan 17;7(1):e29649. doi: 10.1371/journal.pone.0029649

Figure 1. ONOO− nitrates PA700/S10B and increases 26S proteasome activity both in vitro and in intact cell.

Figure 1

In vitro (A–C): ONOO− (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (D–E): HUVEC was incubated with ONOO− for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (in vitro) was subjected to (A) Western blot to detect levels of PA700/S10B and the tyrosine nitration of 26S proteasome/PA700/S10B, (B) 26S proteasome activity (chymotrypsin-like activity), (C) an alternative 26S proteasome activity assay: a substrate-in-gel assay with a fluorogenic substrate followed by fluorescence capturing under the UV light. HUVEC cell lysate was subjected to (D) Western blotting of PA700/S10B tyrosine nitration and (E) assay of 26S proteasome activity (chymotrypsin-like activity). All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.