Figure 5. The pivotal role of p62 PB1 domain in CNOT2 induced degradation of p62/SQSTM1 and the effect of autophagy inhibitors on sub G1 population.

(A) HEK293 QBI cells were co-transfected with HA-CNOT2 and/or wild-type p62 (Myc-p62^wt), p62 PB1 domain mutant (Myc-p62^ΔPB1), p62 LIR/KIR domain mutant (Myc-p62^ΔLIR/KIR) and p62 UBA domain mutant (Myc-p62^ΔUBA) plasmids. Then the cell lysates were immunoprecipitated with HA antibody and subjected to Western blotting with Myc and HA antibodies. (B) The crucial role of p62 PB1 domain in HA-CNOT2 induced p62/SQSTM1 degradation in H1299 cells. H1299 cells were co-transfected with HA-CNOT2 and Myc-p62^wt or Myc-p62^ΔPB1 plasmid and the cell lysates were subjected to Western blotting. (C) The prediction scores for PPI between p62/SQSTM1, ATG5 and LC3B regulated by CNOT2. The scores were represented by STRING using KEGG datasets, containing experimentally verified and putative PPIs. –, known interactions experimentally determined; –, Text Mining; --, suggested interaction in this study [37].