Effect of urate on Nrf2 activation in SH-SY5Y cells.

<p>(<b>a, b</b>) Immunoblotting and reserve transcription PCR analysis showing the protein and mRNA levels of Nrf2, Keap1, γGCLC and HO-1.Cells were treated with 200 µmol/l urate for 0.5, 3, 6 and 14 hours. <i>β</i>-actin served as loading controls. (<b>c</b>) Effect of urate on Nrf2 protein level in the presence of 1 µg/ml CHX. Cells were harvested and lysed at 0, 10 and 30 min after CHX addition with or without urate treatment. (<b>d</b>) Urate did not disrupt Keap1-Nrf2 complex. Cells were treated with 200 µmol/l urate for 6 h. The association of this complex was assessed using IP with anti-Keap1, followed by immunoblotting with anti-Nrf2. (<b>e</b>) Urate inhibited Nrf2 ubiquitination. Cells were treated with or without urate for 6 h in the presence of MG132 (25 µM). For detecting ubiquitinated Nrf2, samples were subjected to IP with anti-Nrf2, followed by IB with an anti-His-HRP-conjugated antibody. The results were independently repeated at least three times.