Extended Data Fig. 1: RTCB-PYROXD1 complex formation and spectroscopic analysis of NADH turnover by PYROXD1.
(a) Co-precipitation of PYROXD1 by immobilized StrepII-GFP-RTCB in the presence of increasing concentrations of NADH. Strep-Tactin beads were washed to remove unbound PYROXD1, and bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. This experiment was repeated three times with similar results. (b) In vitro pull-down experiment with PYROXD1 mutants and StrepII-GFP-RTCB in the presence of divalent metal ions. Strep-Tactin beads were washed to remove unbound PYROXD1, and bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. This experiment was repeated three times with similar results. (c) Spectroscopic analysis of NADH oxidation by human PYROXD1. Data points represent the mean ± SEM of three independent replicates. Solid lines represent a single-exponential fit. (d) In vitro pull-down experiment with recombinant PYROXD1 mutants and immobilized StrepII-GFP-RTCB in the presence of NADH. Strep-Tactin beads were washed to remove unbound PYROXD1, and bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. This experiment was repeated three times with similar results.
