Fig. 3. CST1 interacts with GPX4 to improve the stability of GPX4 protein.

A Schematic of CST1 interactor discovery. The potential interactors were presented at higher levels in the CST1 experimental group than in the IgG control group. Three replicates of IP-MS were conducted. B Silver staining of IP cell lysates. C Venn diagram of three times of LC-MS results showing 63 co-upregulated proteins. D KEGG pathways analysis of 63 co-upregulated proteins. E Immunoprecipitation of the CST1 protein by an anti-GPX4 antibody in MKN45 cells. IgG was used as a negative control. F Immunoprecipitation of the GPX4 protein by an anti-Flag antibody in HEK293 cells transfected with pcDNA3.1-FLAG-CST1. PcDNA3.1-vector was used as a negative control. G Western blot showing GPX4 expression in HGC-27 and MKN45 stable cell lines; total GAPDH was used as a loading control. H, I Degradation of the GPX4 protein was measured after the treatment of 200 µg/ml CHX at the indicated time points in HEK293T, which transfected with Flag-tagged CST1 expression plasmids and Myc-tagged GPX4 expression plasmids. J, K Western blot analysis of GPX4 expression after treatment with 20 µM MG132 for 4 h in MKN45 stable cell lines. Data were expressed as a fold-change relative to control. L Analysis of GPX4 ubiquitination was performed by immunoprecipitation using an anti-Myc antibody, followed by immunoblot with anti-HA antibody and anti-Myc antibody in HEK293T cells transfected with the indicated constructs.