Effect of SKI-II on Keap1.

<p><b>A.</b> Nuclear fractions from BEAS2B cells treated with SKI-II (1 µM) at increasing times (10–120 min) were analysed for Keap1 and Nrf2 expression and normalized using TBP (nuclear). Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p<0.0001, ** p<0.001, * p<0.05. <b>B.</b> Cells were treated with cycloheximide (CXM) and SKI-II (1 µM) at different time points (1 to 24 h) and whole cell extracts were analysed for Keap1 expression normalized against β-actin. Keap1 bands at 140 kDa and 69 kDa were analysed as fold change over non-treatment of the 69 kDa band. *** p<0.0001 when CXM vs. CXM with SKI-II were compared. <b>C.</b> Whole-cell extracts from BEAS2B cells that were treated with SKI-II (1 µM) for 2 and 4 h were immunoprecipitated (IP) using a Keap1 antibody. IP Keap1 was analysed by immunoblotting for ubiquitin modification, p62 and Keap1 expression. IgG: Mouse immunoglobulin control (no cell lysates). Ub: ubiquitin. Hmw: High molecular weight. <b>D.</b> Whole-cell extracts from BEAS2B cells that were pre-treated with arachidonic acid (AA; 10 µM) or biotynilated arachidonic acid (AA-Bio; 10 µM) for 30 min were treated with SKI-II (1 µM) for 2.5 h and immunoprecipitated using Neutravidin Agarose Resin (IP-NA). IP-NA was analysed by immunoblotting for Keap1 expression. <b>E.</b> Whole-cell extracts from BEAS2B cells treated with SKI-II (SK; 1 µM), sulforaphane (SF; 5 µM) or CDDO-Imidazolide (CD; 50 nM) for 4 h, 8 h and 24 h were analysed by immunoblotting for Nrf2, Keap1, NQO1, HO-1 and β-actin. Results are representative of 3 independent experiments and are means and S.E. of triplicates.