Fig. 3

ROS disrupt LDHA tetramer formation and promote noncanonical enzyme activity. a, b ROS disrupt LDHA tetramer formation. HaCaT and HT-3 cell extracts with or without 10 μM H₂O₂ treatment were crosslinked by 0.025 % glutaraldehyde and analyzed by western blotting using LDHA antibody. Tetrameric, dimeric, and monomeric LDHA were indicated (a). HT-3 cell extracts were prepared from 1 × 10⁷ cells with or without 10 μM H₂O₂ treatment and passed over the gel filtration column. Fractions were collected every 0.25 ml per tube and analyzed by western blot for LDHA protein. Molecular mass, 158 and 43 kDa marked below the blots, were determined by Gel Filtration Calibration Kit HMW (GE Healthcare). The loading inputs for gel filtration were shown below (b). c Dimer LDHA presents the noncanonical enzyme activity. The canonical and noncanonical LDHA enzyme activity assays were measured on tetramer fractions (Fraction #56, #57) and dimer fractions (Fraction #60, #61) separated from gel filtration. d, e More dimers form in the LDHA^NLS group compare with that of LDHA^NES group. Cell extracts from HEK293T LDHA KO cells expressing Flag-tagged WT, NLS, and NES LDHA were crosslinked by 0.025 % glutaraldehyde and analyzed by western blotting using LDHA antibody. Tetrameric, dimeric, and monomeric LDHA were indicated (d). Cell extracts from HEK293T LDHA KO cells expressing Flag-tagged WT, NLS, and NES LDHA were passed over the gel filtration column. Fractions were collected every 0.25 ml per tube and analyzed by western blot for LDHA protein. Molecular mass was determined by Gel Filtration Calibration Kit HMW (GE Healthcare). Results are representative of three independent experiments. All data are shown as mean ± SEM. The p values were determined by two-tailed t-test. The values of p < 0.05 were considered statistically significant. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively. NS means non significant