Fig. 2. Ablation of GTPBP8 causes an OxPhos deficiency.

A Western blotting to confirm GTPBP8 knock-out. Whole-cell lysates from HEK293, GTPBP8^KO1 and GTPBP8^KO2 were used for SDS-PAGE analysis. GTPBP8 antibody was used to detect GTPBP8 endogenous levels; SDHA antibody was used as a loading control. B Growth rates of GTPBP8 knock-out cells in galactose medium. HEK293 (black), GTPBP8^KO1 (red), and GTPBP8^KO2 (yellow) were cultured in DMEM containing 0.9 g/l galactose (n = 3 independent experiments). The mean average cell number per each time point is indicated, and error bars = ± 1 standard deviation (SD). Statistical analyses were performed using the Student’s two-tailed t-test. P-values and source data are provided in the Source Data file. C Western blotting to assess OxPhos complexes steady-state levels in GTPBP8 knock-out cell lines. Whole-cell lysates from HEK293, GTPBP8^KO1 and GTPBP8^KO2 were resolved via SDS-PAGE. Membranes were probed using the OxPhos antibody cocktail against nuclear-encoded proteins of complexes V (ATP5A) and I (NDUFB8) and mtDNA-encoded complex IV protein MTCO2. GAPDH was used as a loading control. D LFQ mass spectrometry analyses of OxPhos complexes in GTPBP8 knock-out cell lines compared to HEK293 (n = 3 independent experiments). Log₂(FC) of GTPBP8^KO1 vs HEK293 (red) and GTPBP8^KO2 vs HEK293 (yellow) is represented on the y-axis for each OxPhos complex. Each dot refers to a protein, which constitutes the specific complex. A summary of boxplots is provided in a Source Data file. All the log₂(FC) values are listed in Supplementary Data 3. E Assessment of OxPhos complex I and complex IV activities in GTPBP8 knock-out cell lines compared to control. Mitochondrial lysates from HEK293, GTPBP8^KO1 and GTPBP8^KO2 cell lines were resolved by BN-PAGE and stained using complex I (CI) and complex IV (CIV) -specific substrates (n = 1 independent experiment). Coomassie staining was used as a loading control. SC supercomplexes. Source data are provided as a Source Data file.