Fig 1: CSE induces p62 oligomers via their Keap1 interaction region (KIR). (A) BEAS-2B cells were incubated with HC-CSE for 24 hours and p62 was immunoprecipitated (IP) from whole-cell extracts. IP-p62 species were resolved by SDS-PAGE followed by Western blot (WB) and membranes immune-stained against ubiquitin (Ub), LC3, and p62. IgG control (no whole-cell lysates used). (B) Molecular weight characterization of p62 oligomers. HEK293 cells were transfected with p62-FLAG and incubated with HC-CSE for 24 h. Whole-cell extracts were resolved by WB and immune-stained against FLAG. A molecular marker for high molecular weights (MW) was used. A graph extrapolating distance of bands from origin and MW determined the expected size of p62 oligomers and the possible combinations. (C) Deubiquitination assay of IP-p62. BEAS-2B cells were prepared as indicated in (A). Samples were divided by two and p62 was IP. IP-p62 beads were incubated in deubiquitin buffer plus/minus Ubiquitin specific peptidase 2 (USP2). (D) Schematic representation of p62 functional domains: PB1, ZZ (ZZ-type zinc finger domain), LIR, KIR, and UBA and schematic representation of the different deletion p62 mutants used in (E). (E) HEK293 cells were transfected with either WT p62-GFP, or the following p62-GFP deletion plasmids: ?123-170, ?170-256, ?256-370, ?346-385, and ?UBA (?386-440) and incubated with HC-CSE for another 24 h. WB was performed and membranes were immune-stained using anti-GFP and ß-actin. (F) BEAS-2B cells were transfected with siRNA for Keap1 or a random oligonucleotide control (NC) and incubated with LC- and HC-CSE for a further 24 h. WB was performed and membranes were immune-stained using against p62, LC3, Keap1, and ß-actin. (G) HEK293 cells were transfected with either WT p62-GFP or a KIR deletion p62-GFP plasmid and incubated with HC-CSE for another 24 h. WB was performed and membranes were immune-stained using anti-GFP and ß-actin. Data are representative of at least two independent experiments