PARK7 is oxidized by TNFSF10 and the oxidation of PARK7 is required for the interaction of R-HSPA5 and SQSTM1 in HCT116 cells treated with TNFSF10. (a) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h, followed by immunoblotting with specific antibodies for oxidized PARK7 and PARK7. (b) HCT116 cells were treated with 10 ng/ml TNFSF10 for 4 h or co-treated with 10 ng/ml TNFSF10 and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (c) HCT116 cells were treated with 250 μM tert-butyl hydroperoxide (tBHP) for 3 h or co-treated with 250 μM tBHP and 2.5 mM NAC, followed by immunoblotting with oxidized PARK7 antibody. (d) HCT116 cells were co-transfected with plasmids encoding Ub-R-HSPA5-GFP and one of the following: FLAG-tagged wild-type PARK7 or its C46A, C53A, and C106A mutants. After 48 h, the cells were treated with 10 ng/ml TNFSF10 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-GFP or anti-FLAG antibody. (e) HCT116 cells were transfected with a plasmid encoding FLAG, FLAG-PARK7, or FLAG-PARK7 C106A . After 48 h, cells were treated with 20 ng/ml TNFSF10 for 2 h. Cell lysates were immunoprecipitated with the anti-FLAG antibody and then immunoblotted with the indicated antibodies.
{}