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. Author manuscript; available in PMC: 2021 Apr 15.
Published in final edited form as: Cell Rep. 2021 Mar 16;34(11):108869. doi: 10.1016/j.celrep.2021.108869

Figure 3. Absence of SFXN1 leads to complex-III-related defects.

Figure 3.

(A) Steady-state abundance of select mitochondrial proteins, including OXPHOS components, subunits of import machineries, and other proteins in the IMM and matrix.

(B) Immunoblotting for Fe-S containing subunits of respiratory complexes in mitochondrial extracts. GRP75 served as loading control.

(C) Immunoblotting for mtDNA-encoded subunits in mitochondrial extracts. GRP75 served as loading control.

(D) Densitometric analysis of bands for select proteins in (A), (B), and (C). Protein levels in WT were set to 1.0 (mean ± SEM, n ≥ 4).

(E) Electron flow through the respiratory chain in intact mitochondria. Base buffer contains 10 mM pyruvate, 2 mM malate, and 4 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Injections of complex inhibitors/substrates were performed as indicated. rot, rotenone; succ, succinate; AA, antimycin A; asc/TMPD, ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine.

(F) Spectrophotometric assays using detergent-solubilized mitochondria to monitor individual complex activities. CI, oxidation of NADH to NAD+; CII, ubiquinol production; CIII, cytochrome c reduction; CIV, cytochrome c oxidation (mean ± SEM, n ≥ 4).

(G) 1D BN assembly. Mitochondria solubilized in 1% (w/v) digitonin were resolved on a 4%–16% BN gel and immunoblotted for the indicated subunits.

(H) Mitochondria solubilized in 1% (w/v) digitonin were resolved by 2D BN/SDS-PAGE and immunoblotted for the indicated subunits. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; unpaired Student’s t test.