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Plates were subsequently washed with 0.1% polyethylene sorbitan monolaurate (Tween20)-Tris-buffered saline (TTBS), immediately fixed with cold 3.7% paraformaldehyde in PBS, and re-treated with the blocking solution overnight at 4C

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Blots were incubated overnight at 4 C with HRP-conjugated anti-nitrotyrosine mouse mAb (1:2500 dilution in CanGetSignal solution 1,NKB-101, Toyobo, Osaka, Japan), followed by ECL-detection on a FUSION SOLO.7S.EDGE imaging system (Vilber Lourmat, Marne-la-Valle, France)