Fig 2: Grx2 deficiency is associated with dysregulation of the disulfide relay system. (A) Genes involved in the disulfide relay system were correlated to Grx2 using 42 BXD mouse strains fed a chow diet. Grx2 is positively correlated with Gfer and Cox17 in skeletal muscle. (B) Tibialis anterior muscle protein was extracted from saline and colchicine injected mice, and protein expression level of GFER was lower in Grx2-/- skeletal muscle, as determined by western blot. (C) Protein expression of COX17 was increased in Grx2-/-, and decreased in skeletal muscle from mice treated with colchicine. Equal protein loading was determined with Ponceau stain. (D) Complex IV activity is decreased in skeletal muscle from Grx2-/- mice. Data are expressed as mean ± SEM. n = 6–7 for GFER and COX17 quantification; n = 6 for complex IV activity. *p < 0.05.

Fig 3: Western blot analysis of mitochondrial proteins. Twenty micrograms of total cellular proteins from various cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for (A) 8 mtDNA-encoding (ND1, ND3, ND4, ND4L, ND5, CYTB, CO2 and ATP8) and (B) 10 nucleus-encoding (NDUFA1, NDUFS1, NDUFA10, SDHB, UQCRC2, UQCRFS1, COXIV, COX17, ATP5B and ATP5F1) subunits of OXPHOS (CI, CII, CIII, CIV and CV represented complexes I, II, III, IV and V, respetively), TOM20 or GAPDH as a loading control, respectively. (C) Quantification of mitochondrial protein levels. Average relative ND1, ND3, ND4, ND4L, ND5, CYTB, CO2, and ATP8 content per cell, normalized to the average content per cell of TOM20 in TRUB1KO and WT cell lines. The values for the TRUB1KO cell lines are expressed as percentages of the values for the WT cell line. The error bars indicate two standard errors of the means, the horizontal dashed lines represent the average value for each group. The calculations were based on three independent determinations in each cell line. P indicates the significance, according to the t-test, of the differences between TRUB1KO and WT cell lines.