Fig. 2. OTUD5 is a GPX4-interacting protein for stabilization in response to I/R.

a PRTCs were induced by H/R in the presence or absence of the proteasome inhibitor MG132, or the lysosome inhibitor chloroquine (CQ) for 3 h. Cells were collected, and the protein levels were analyzed by immunoblotting. b Immunoblot and quantification analysis of GPX4’s ubiquitination in PRTCs upon H/R induction. c The protein lysate of HK2 cells was combined with an anti-human GPX4 antibody or the isotype IgG for immunoprecipitation. The number of GPX4 interacting proteins in the protein complex was identified using LC–MS/MS. d Immunoblot and quantification analysis of GPX4 expression in HK2 cells infected with siOTUB1 or siOTUD5, respectively. e HK2 cells were treated with H/R for 3 h, and the cell lysates were mixed with an anti-human GPX4 or anti-human OTUD5 antibody for immunoprecipitation. The immunoprecipitated protein complex was collected, and protein levels were detected by immunoblotting. f Representative fluorescence images of GPX4 and OTUD5 expression in cells treated with or without H/R for 3 h. Scale bar = 50 or 10 μm. g Immunoblotting and quantification of GPX4 and OTUD5 expression, and GPX4’s ubiquitination in siOTUD5-infected HK2 cells treated with or without H/R for 3 h. h Immunoblotting and quantification of GPX4, OTUD5, and GPX4’s ubiquitination in His-tagged OTUD5 plasmid-infected HK2 cells treated with or without H/R for 3 h. i Immunoblotting and quantification of GPX4 and OTUD5 expression in HK2 cells infected with WT OTUD5 or C224S OTUD5 and subjected to H/R for 3 h. j Immunoblotting and quantification of GPX4 and OTUD5 expression in siOTUD5-infected HK2 cells, which were pretreated with Cycloheximide (CHX) for 1, 2, or 4 h and subjected to H/R for 3 h.