Figure 7: An α-helical peptide of p62 interrupts the TRB3/p62 interaction and promotes autophagy and UPS degradation.
(a) HepG2 cells were treated with Pep2–A2 or Pep2-con (5 μM) for 12 h, and extracts were IP with anti-p62 Ab or normal rabbit IgG and blotted with anti-TRB3 Ab. (b,c) HEK 293T cells were transfected with the indicated plasmids. The effect of Pep2–A2 on the LC3/p62 and Ub/p62 binding was evaluated with Co-IP assay. (d) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h, and the colocalization of the P62/TRB3 was examined by immunostaining. Scale bar, 10 μm. (e) HepG2 cells were treated with Pep2-con, Pep2–A2 or Pep2–A2mut (5 μM) for 12 h and cell extracts were IP with anti-p62 Ab and blotted with anti-TRB3 Ab. (f) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h and the autophagy-associated proteins were detected by immunoblotting. (g) HepG2 cells infected with mRFP-GFP-LC3 plasmid were treated with Pep2–A2 or Pep2-con for 24 h, and the images were captured with confocal microscopy. Scale bar, 10 μm. See also Supplementary Movies 4–6 for details. (h) HepG2 cells transfected with UbG76V-GFP plasmid were treated with Pep2–A2 or Pep2-con for 24 h. Cell lysates were collected for immunoblotting analysis. (i) HepG2 cells were treated with Pep2–A2 or Pep2-con for 24 h. The expressions of indicated proteins were detected by immunoblotting. (j) HepG2 cells were treated with Pep2-con, Pep2–A2, Pep2–A2 plus bafilomycin (left) or Pep2–A2 plus MG132 (right) for 12 h. The expression of indicated proteins was detected by immunoblotting. For all panels, n=3 independent experiments. Data indicate mean±s.e.m. Statistical significance was determined with one-way ANOVA; **P<0.01.
