Figure 4 : ATM phosphorylates PEX5 at Ser 141 in response to ROS.

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

Figure 4

(a) Immunoprecipitation of lysates from HEK293 cells overexpressing Myc–PEX5 and treated with H2O2 (0.4 mM) for 1 h, using an anti-Myc antibody followed by immunoblotting with a phospho-(S/T) ATM substrate antibody. Inputs were immunoblotted using the indicated antibodies. (b) Immunoprecipitation performed with anti-Myc antibody of lysates from HEK293 cells overexpressing Myc–PEX5, treated with H2O2 (0.4 mM) for 1 h in the presence/absence of an ATM inhibitor (KU-55933, 2 h pretreatment) followed by immunoblotting with phospho-(S/T) ATM substrate antibody. Inputs were immunoblotted using the indicated antibodies. (c) HEK293 cells transfected with either a Myc–PEX5-WT or Myc–PEX5-S141A mutant construct treated with H2O2 (0.4 mM) for the indicated times. Immunoprecipitation was performed with an anti-Myc antibody followed by immunoblotting with phospho-(S/T) ATM substrate antibody. Inputs were immunoblotted using the indicated antibodies. (d) HEK293 cells transfected with either a Myc–PEX5-WT or Myc–PEX5-S141A mutant construct treated with H2O2 (0.4 mM) for 1 h. Western analysis was performed with anti-pPEX5 (Ser 141), Myc, pATM (Ser 1981), ATM and GAPDH antibodies. (e) HEK293 cells transfected with Myc–PEX5-WT for 24 h following a siRNA knockdown of ATM for 48 h treated with H2O2 (0.4 mM) for 1 h. Western analysis was performed with anti-pPEX5 (Ser 141), Myc, pATM (Ser 1981), ATM and GAPDH antibodies. (f) AT (GM05849) fibroblast cells were transfected with Myc–Pex5 and Flag–ATM-WT or Flag–ATM-RQ mutant for 48 h and treated with H2O2 (0.4 mM) for 1 h. Western analysis was performed with anti-pPEX5 (Ser 141), Myc, pATM (Ser 1981), ATM and GAPDH antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 9.