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. 2022 Dec 31;299(2):102857. doi: 10.1016/j.jbc.2022.102857

Figure 3.

Figure 3

H2O2regulates SOD1 LLPS associated with Cys111 and Trp32.A, SDS-PAGE of 10 μM EGFP-SOD1 was treated with 0 to 20 mM H2O2 after 2 h at 37 °C. M: monomer, D: dimer. Dashed arrows indicated oxidation upshift strips. Asterisks indicated impure protein. B, SDS-PAGE of 10 μM EGFP-SOD1, C111S, and W32S treated with 0, 1, 20 mM H2O2 after 2 h at 37 °C. M: monomer, D: dimer. Dashed arrows indicated oxidation upshift strips. Asterisks indicated impure protein. C, confocal microscopy images of 20 μM EGFP-SOD1, EGFP-C111S, EGFP-W32S, EGFP-C111S/W32S with or without H2O2 (1 mM, 2 h at room temperature) in the presence of 15% PEG. Scale bar represents 10 μm. D, area quantification of the droplets from (B) via intensity thresholding and region of interest (ROI) auto-selection. Histogram of the average area of all droplets. n = 3 biologically independent samples. Data are presented as mean values ± S.D. Data were analyzed by two-way ANOVA with multiple comparisons. ns, not significant. E, confocal microscopy images of 40 μM EGFP-SOD1, EGFP-C111S, EGFP-W32S, EGFP-C111S/W32S treated with H2O2 (0, 1, 20 mM; 10% PEG; 37 °C for 2 h). Scale bar represents 10 μm. LLPS, liquid-liquid phase separation; SOD, superoxide dismutase.