Figure 3. Role of p62 on sequestration of ubiquitin and I-κBα.

Su-DHL8 cells were pre-incubated with or without 50 µM CQ for 1 hour and then treated with indicated concentration of bortezomib for 24 hours. (A) Co-localization of ubiquitin and p62. Cells on slides were co-stained with monoclonal anti-ubiquitin (1∶20 dilution) and polyclonal anti-p62 (1∶100 dilution) antibodies, and probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution) antibodies. (B) Co-localization of p62 and LC3-II. Cells on slides were co-stained with monoclonal anti-p62 (1∶20 dilution) and polyclonal anti-LC3B (1∶100 dilution), and probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution) antibodies. (C) Bortezomib-induced ubiquitination of I-κBα and accumulation of LC3-II protein. After treatment with 20 nM bortezomib with or without CQ, proteins were extracted from Su-DHL8 cells with 1% SDS and loaded onto 4–20% NuPAGE. Polyclonal anti-IκBα (1∶200) and monoclonal anti-ubiquitin (1∶200) were used to probe specific proteins. (D) Co-localization of p62 and I-κBα. Cells on slides were co-stained with polyclonal anti-I-κBα antibody (1∶20) and monoclonal anti-p62 antibody (1∶20) and then probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution). Arrows indicate p62-I-κBα aggregates. (E) Detection of p62 and I-κBα interaction by Co-IP. Polyclonal anti-I-κBα antibody (2 µg) or normal rabbit IgG was coated onto Dynabeads protein A and 300 µg proteins were used for IP. Monoclonal anti-p62 antibody (1∶100 dilution) and polyclonal anti-I-κBα antibody were used to detect p62/I-κBα protein complex.