Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease

doi: 10.1038/gim.2017.251

Figure Lengend Snippet: (a-b) In skeletal muscle immunoblotting detected decreased steady-state levels of mitochondrial OXPHOS complex subunits for complexes I, III, IV and V. (c-d) BN-PAGE detected reduced complexes I, IV and V. (e) Immunoblotting detected slightly reduced SLC25A21 in the patient`s fibroblasts, while (f) mitochondrial OXPHOS proteins were normal. (g) BN-PAGE detected normal respiratory chain complexes in the patient`s cell line. Nuclear-encoded GAPDH, VDAC, SDHA or SDHB proteins were used as loading controls. Abbreviations: Complex I (CI); complex II (CII); complex III (CIII); complex IV (CIV); complex V / F 1 F O ATP synthase (CV); (GAPDH); cytochrome c oxidase 1 (COX1); cytochrome c oxidase 2 (COX2); voltage-dependent anion-selective channel (VDAC); NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NDUFB8); F 1 F O ATP synthase subunit (ATP5A); succinate dehydrogenase subunit A (SDHA); succinate dehydrogenase subunit B (SDHB); ubiquinol-cytochrome c reductase core protein II (UQCRC2).

Article Snippet: A codon-optimised SLC25A21 gene was synthesized by GenScript (Piscataway, USA), and cloned into the expression vector pNZ8048 under the control of a nisin A-inducible promoter .

Techniques: Western Blot

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease

doi: 10.1038/gim.2017.251

Figure Lengend Snippet: ( a ) Membrane view of the comparative homology model of human SLC25A21 generated with SwissModel (Arnold et al., 2006) based on the structure of the bovine ADP/ATP carrier (alignment shown in ). The matrix and cytoplasmic salt bridge network are shown in dark blue and cyan, respectively. The mutated lysine 232 is shown in yellow. The residues of the proposed substrate binding site are shown in green and the substrate 2-oxoglutarate is shown in an orange ball and stick representation. ( b ) Cytoplasmic view showing the residues involved in the formation of the matrix salt bridge network in dark blue. The most probable side chain conformations of the p.(Lys232Arg) mutation are shown in magenta, indicating that the substitution might impair binding to Glu131 and might lead to other polar interactions. ( c ) The 2-oxoglutarate uptake curves of SLC25A21 and SLC25A21_p.(Lys232Arg). Fused membrane vesicles of Lactococcus lactis expressing SLC25A21 (triangles) or SLC25A21_p.(Lys232Arg) (circles) were pre-loaded with 5 mM 2-oxoglutarate and transport was initiated with the external addition of 1.5 μM [ 14 C]-2-oxoglutarate. Membranes of the uninduced SLC25A21 strain (squares) show background binding, indicating that SLC25A21_p.(Lys232Arg) does not transport above background. ( d ) Expression levels of the wild-type and mutant SLC25A21 were similar, indicating the mutation did not affect expression and insertion of the carrier in lactococcal membranes. ( e ) The 2-oxoglutarate transport rates of SLC25A21 and SLC25A21_p.(Lys232Arg) after correction for background binding. The error bars represent the standard deviation of four technical repeats. Student t-tests, significant uptake above background: P > 0.05, not significant (NS); P > 0.01, *; P > 0.001, **; P > 0.0001, ***; P < 0.0001, ****.

Article Snippet: A codon-optimised SLC25A21 gene was synthesized by GenScript (Piscataway, USA), and cloned into the expression vector pNZ8048 under the control of a nisin A-inducible promoter .

Techniques: Membrane, Generated, Binding Assay, Mutagenesis, Expressing, Standard Deviation