Fig. 2.

Two conserved cysteine residues located in a disordered region of p62 are required for formation of DLC and ubiquitylated aggregates. a Alignment showing cysteines 105 and 113 (highlighted) are conserved in vertebrates. Increasing conservation across species is shown by light-to-dark red. b p62^−/− MEFs stably expressing either wild type or C105A,C113A FLAG-p62 were treated with H₂O₂ (3 mM, 1 min) or PR-619 (5 μM, 10 min) and immunoblotted in non-reducing conditions. c Formation of ubiquitylated, p62-positive aggregates in stable cells described in b following oxidative stress (1 mM H₂O₂ or 5 μM PR-619 for 30 min). Cells were immunostained for ubiquitin and p62, analysed by confocal microscopy (c) and quantified (d). Error bars represent s.e.m., n = 3, **P < 0.01, ***P < 0.005 (unpaired t-tests). Arrows indicate the positions of monomeric and oligomeric p62. Scale bar: 10 µm