Fig. 5: 13-HODE directly bound to catalase (CAT) and inhibited CAT activity.

a Primary mouse hepatocyte proteins were pulled down by biotin-labeled 13(S)-HODE followed by proteomics; (b) top 10 proteins based on the Vina scores from in silico molecular docking; (c) structural overview of CAT monomer-13(S)-HODE and CAT tetramer-13(S)-HODE model. d Surface plasmon resonance assay for interaction of 13(S)-HODE was passed over the Biacore chip surfaces immobilized with recombinant CAT protein: Biacore diagram and estimated dissociation constant value (KD) for 13(S)-HODE binding CAT. e Western blot analysis of the CAT protein levels in 13-HODE treated hepatocytes; n = 5 independent experiments. f CAT activity of hepatocytes treated with 13-HODE (1 μM) for 2, 6, or 12 h; n = 5 independent experiments. g Recombinant CAT protein was incubated with 13-HODE for 20 min, and CAT activity was measured; data are from 3 repeats. h CAT activity in 12-month-old or 2.5-month-old mouse livers; n = 6 mice per group. i ROS level demonstrated by DCFH-DA and fluorescence analysis of primary mouse hepatocytes treated with 13-HODE; scale bar = 250 μm; n = 5 independent experiments. j primary mouse hepatocytes were treated with 1 μM 13-HODE for 6 h; cells were pretreated with 100 μM DSS crosslinker for 1 h before protein extraction: Western blot analysis of protein level of tetramer and monomer of CAT; n = 4 independent experiments. Data are presented as the mean ± SEM. Two-tailed student’s t test was performed for e, f, h, i. Figure 5a was created with Biorender.com.