Figure 4

The transient inhibition of IκBα poly-ubiquitination by H₂O₂. (A) HEK293 cells were incubated with H₂O₂ (0.5 mM) for various times, and the cells were lysed in hypotonic buffer to prepare cell extract. Chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolase (PGPH) peptidase activities were measured using fluorogenic peptide substrates (n = 3). (B) HEK293 cells were incubated with or without MG-132 (20 µM) for 1 h, and stimulated with TNFα (20 ng/ml) for up to 20 min. The cells were lysed, and immunoblotting analysis was performed using the anti-IκBα antibody. (C) Cells were incubated with MG-132 for 1 h, and a group of cells was exposed to 0.5 mM H₂O₂ for 20 min. After stimulation with TNFα, cells were lysed at various times. The cell lysate was analyzed by immunoblotting using anti-IκBα (upper panel), anti-ubiquitin (middle panel) and anti-β-actin (lower panel) antibodies.