Fig 1: Macrophage LPS stimulation destabilizes Complex II.(A) Illustration of the five major respiratory complexes. Immortalized bone marrow–derived macrophages (iBMDMs) were synchronously stimulated with out without LPS (200 ng/ml) for 8 or 24 hours, and SDHA or control immunoglobulin G (IgG) was immunoprecipitated. SDS–polyacrylamide gel electrophoresis (PAGE) and IB analysis of ATP5A (Complex V adenosine 5´-triphosphate synthase F1 subunit alpha), UQCRC2 (ubiquinone Complex III cytochrome b-c1 complex subunit 2), SDHA, SHDB, and NDUFB8 (Complex I NADH dehydrogenase ubiquinone beta subcomplex subunit 8). (B) Quantification of SDHA and SDHB subunit abundance relative to ATP5A signal. (C) Quantification of immunoprecipitated SDHB relative to SDHA. Fluorescence measurements are reported in arbitrary units (A.U.). (D) Blue native (BN)–PAGE and SDHA IB analysis from iBMDM stimulated with or withoutLPS for 8 or 24 hours. Intensity profile of the SDHA blot showing Complex II–associated SDHA (~200 kDa) and dissociated SDHA (~100 to 70 kDa). (E) Percentage of SDHA in Complex II and dissociated SDHA populations. (F) Clear native (CN)–PAGE and SDH in-gel activity assay from iBMDM stimulated with out without LPS for 4, 8, or 24 hours. (G) SDH in-gel activity assay normalized to sample protein content. (H) Flow cytometric analysis of SDHB levels in total, CD3+, CD19+, and CD11b+ splenocytes from mice injected intraperitoneally (I.P.) with LPS (20 mg/kg) or phosphate-buffered saline (PBS). The mean fluorescence intensity (MFI) within each subset was plotted as a fold difference compared to the PBS-injected total splenocyte MFI. SDS-PAGE and IB analysis of SDHA, SDHB, IRG1 (immune-responsive gene 1), and iNOS (inducible NO synthase) from Nos2-/- (I) and Acod1-/- (J) BMDM stimulated with or without LPS for 24 hours. Graphs presented as the mean of n = 3 independent experiments with SD error bars. P values were calculated using an unpaired t test or one-way analysis of variance (ANOVA) with Tukey’s posttest. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (A) was adapted from BioRender.com assets. co-IP, coimmunoprecipitation; MW, molecular weight; ns, not significant; WT, wild type.