Cys374 is required for actin glutathionylation, actin polymerization, and NET formation. (A) Glutathionylated proteins in activated human neutrophil lysates were immunoprecipitated with anti–GSH antibody (time-dependent after C5a stimulation) and immunoblotted by using anti–pan-actin antibody. A total cell lysates (Input) was loaded as control. Data are representative of five independent experiments. (B) Immunoblotting. Actin glutathionylation in activated Hoxb8 mouse neutrophils overexpressing EGFP-β-actin-Cys³⁷⁴ (WT), point-mutated Cys³⁷⁴ to Ala³⁷⁴, or Cys³⁷⁴ to Glu³⁷⁴ was detected by anti–GSH antibody. The overexpression of WT and mutant forms of EGFP-β-actin was confirmed by immunoblotting with anti–GFP antibody. The ratio of glutathionylated EGFP-β-actin to total EGFP-β-actin was calculated as actin-glutathionylation in the bar graph (right panel). (C) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys³⁷⁴ (WT), point-mutated Cys³⁷⁴ to Ala³⁷⁴, and Cys³⁷⁴ to Glu³⁷⁴ mouse neutrophils with the indicated triggers. Right: Quantification of F-actin polymerization. (D) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of WT and overexpressed EGFP-β-actin-Cys³⁷⁴ (WT), point-mutated Cys³⁷⁴ to Ala³⁷⁴, and Cys³⁷⁴ to Glu³⁷⁴ mouse neutrophils with the indicated triggers. Right: Quantification of released dsDNA in supernatants of activated neutrophils. Data are means ± SEM. ***, P < 0.001. n = 3. Bars, 10 µm.