Figure 4A. Interaction of GPx8 with human caspase-4 (Casp4) or mouse caspase-11 (Casp11) was confirmed by co-IP assays. 293T cells were co-transfected with vectors expressing FLAG tagged Casp4 or Casp11, in addition to GPx8 expressing vectors. Cell lysate containing Casp4- or Casp11-FLAG proteins was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 and anti-FLAG Ab (left panel). GPx8 and its interacting proteins were precipitated by anti-GPx8 Ab and analyzed by the indicated Abs (right panel).B. Co-localization of Myc-tagged GPx8 (red) and endogenous caspase-4 (green) in THP-1 cells.C. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (F) C79S, C108S, and C2S2 mutants of GPx8 abolished most of the binding to caspase-4 by co-IP assays.C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. THP-1 cells stably expressing empty vector (EV), WT, or mutant GPx8 proteins were confirmed by immunoblotting (G).C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).