Figure 6 : Ubiquitylated PEX5 binds with the autophagy adaptor protein p62 in response to ROS.

From: ATM functions at the peroxisome to induce pexophagy in response to ROS

Figure 6

(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 images of HepG2 cells treated with H2O2 (0.4 mM) for 3 h and immunostained with p62 (green), PEX5 (red) and ubiquitin (purple). Scale bars, 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 bars, 10 μm. High-magnification images of the outlined areas are shown to the right (scale bars, 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 the 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 6 h. Lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-HA antibodies. Inputs were immunoblotted using the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 9.