Specificity of antibody against phosphorylated p62 at serine 349 (S349). (a) Schematic representation of human p62. Number of amino acid is indicated. PB1, proteasomal binding region 1; ZnF, zinc finger; LIR, LC3-interacting region; KIR, Keap1-interacting region; UBA, ubiquitin associated domain. (b) Alignment of the KIR in p62 homologues. Underline represents the epitope for the antibody against phosphorylated p62. (c) Synthetic peptide (344–354 of human p62) was used to confirm the reactivity of phosphorylated p62-specific antibody by dot blot analysis. Phosphorylated peptide (P-peptide) is diluted, applied to a membrane and detected with the antibody against p62 phosphorylated at S349 (P-S349), whereas non-phosphorylated peptide (NP-peptide) is not detected. (d) HeLa cells were treated with an autophagic inhibitor, bafilomycin A (Baf), or a proteasome inhibitor, epoxomicin (Epo), for the indicated times. Two types of phosphorylation-specific antibodies differentially recognise p62 with phosphorylation on distinct residues. The molecular mass is indicated on the left side of the panel. (e, f) Quantitative data represents the ratio of the P-S349 (e) or P-S403 (f) level relative to the total p62 level at the indicated times following Baf or Epo treatment.