Figure 6. Ubiquitinated PEX5 binds with autophagy adaptor protein p62 in response to ROS.

(a) HepG2 cells treated with H₂O₂ (0.4 mM) for the indicated time points were immunoprecipitated with anti-PEX5 and immunoblotted with anti-p62 antibodies. Inputs were immunoblotted using the indicated antibodies. (b) Representative image of HepG2 cells treated with H₂O₂ (0.4 mM) for 3h and immunostained with p62 (green) , PEX5 (red) and ubiquitin (purple). Scale bar, 10 μm. (c) Western analysis of whole cell extracts (WCE) and peroxisome fractions (P) of HepG2 cells treated with 0.4 mM of H₂O₂ for 1 h immunoblotted using anti-p62, anti-PMP70 and catalase antibodies. (d) Representative images of FAO cells treated with 0.4 mM of H₂O₂ for 1 h and immunostained with p62 (green) and PMP70 (peroxisomes-red). Scale bar, 10 μm. High-magnification images of boxed areas are shown to the right (Scale bar, 2.5 μm). (e) Subcellular fractionation of HEK293 cells overexpressing Myc-PEX5 WT or Myc-PEX5 K209R (mutant) treated with 0.4 mM of H₂O₂ for 3 h. Immunoblotting was performed with indicated anti-Myc, catalase, PMP70, phospho-ATM and ATM antibodies. WCE, whole cell extract; P, peroxisome fraction. (f) HEK293 cells transfected with HA-p62 and Myc-PEX5-WT or Myc-PEX5-K209R, and treated with 0.4 mM of H₂O₂ for 6h. Lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-HA antibodies. Inputs were immunoblotted using the indicated antibodies. Uncropped images of western blots are shown in Supplementary Fig. S9.