Fig. 5.

Analyses of proteasome complex upon oxidative stress. (A) Analysis of proteasome complexes under oxidative stress. Wild-type (WT) or KIAA0368-deficient (KO) MEF cells were treated with 100 μM H₂O₂ or H₂O for 20 min. Five hundred micrograms of whole cell lysates were fractionated by glycerol density gradient (10–40% glycerol from 1 to 32 fractions) and immunoblotted with indicated antibodies. Asterisk indicates non-specific bands. W.C.L.; whole cell lysate. (B) Peptidase activities under oxidative stress. Odd fractions prepared in (A) were measured using Suc-LLVY-AMC. The vertical axis represent relative fluorescence normalized to peptidase activity of the first fraction. (C) Detection of oxidatively damaged proteins. Wild type and KIAA0368-deficient MEF cells were treated with 200 μM H₂O₂ for indicated times, then cell lysates were immunoblotted with indicated antibodies. (D) Accumulation of ubiquitylated proteins. Wild-type and KIAA0368-deficient MEF cells were treated with 200 μM H₂O₂ for 120 min in the presence or absence of 20 μM MG132 for 60 min, then the cell lysates were immunoblotted with indicated antibodies.