Fig. 6. OTUB1 C23 and C204 are S-glutathionylation targets that increase GSH in a system XC−–dependent manner.
(A) OTUB1-WT or OTUB1-C23S or OTUB1-C204S (left) and OTUB1-WT, OTUB1-C91S, or OTUB1-C212S (right) were expressed in H522 cells. PSSG was immunoprecipitated using GSH antibody followed by detection of OTUB1-SSG via Western blot analysis. OTUB1 and ACTB from total cell lysates were used as input. (B) Confirmation of equal expression of OTUB1 plasmids used in (C). ACTB, loading control. (C) Assessment of GSH in H522 cells overexpressing 125 ng of OTUB1-WT, OTUB1-C23S, OTUB1-C91S, OTUB1-C204S, and OTUB1-C212S plasmids or pCMV vector control. (D) Western blot analysis of SLC7A11 and OTUB1 in H522 cells transfected with control or SLC7A11 siRNA and WT OTUB1 plasmid or pCMV empty vector control. ACTB, loading control. (E) GSH in H522 cells following knockdown of SLC7A11 and overexpression of WT OTUB1. *P < 0.05, ***P < 0.001, and ****P < 0.0001.