Fig. 7

CrCYB5D1 participates in redox-dependent regulation of coordinated flagellar beating. (A) Assessment of the redox state of CrCYB5D1::Strep in isolated flagella treated with 1 mM H₂O₂ or 1 mM DTT; NT indicates no treatment. (B) Summary of identified disulfide bonds from the samples in A by PRM MS. The number of peptide spectral matches (PSMs) are indicated. (C) Comparison of heme-binding activity of CrCYB5D1::Strep from the flagella of DTT- or H₂O₂-treated cells using hemin-conjugated agarose;. NT indicates no treatment. ?P,? the soluble fraction of the flagella before incubation with hemin-conjugated agarose beads; ?E,? the bound fraction eluted with SDS and ?-mercaptoethanol. (D) Immunoblot analysis of flagellar samples from HS211 and Crcyb5d1 cells treated with 1 mM DTT or 1 mM H₂O₂ using antibody against outer arm dynein LC3; ?-tubulin served as a loading control. (E and F) Percentage of HS211 and Crcyb5d1 cells exhibiting coordinated flagellar beating upon treatment with 1 mM DTT, 40 mM TEMPOL, 0.5 mM t-BOOH, or 2 mM H₂O₂. The number of beat cycles is indicated. All values are shown as the mean ± SEM of three independent experiments. ***P < 0.001. n.s., not significant.