Journal: Molecular Genetics & Genomic Medicine
Article Title: Mutations in the mitochondrial tryptophanyl‐tRNA synthetase cause growth retardation and progressive leukoencephalopathy
doi: 10.1002/mgg3.654
Figure Lengend Snippet: (a) Left: representative acidic‐UREA PAGE followed by Northern blot analysis indicates the aminoacylated (Trp‐tRNA Trp , Ser‐tRNA Ser ), versus uncharged (tRNA Trp , tRNA Ser ) tRNA under acidic (pH < 5) or basic (OH‐) conditions in fibroblasts of subjects (II:1 and II:2) and one nonrelated control (C1). Right: quantification of (a) under acidic (pH < 5) conditions. (B) Left: representative acid‐urea PAGE followed by Northern blot analysis indicates the aminoacylated (Trp‐tRNA Trp , Ser‐tRNA Ser ) versus uncharged (tRNA Trp , tRNA Ser ) tRNA in acidic (pH < 5) or basic (OH ‐ ) conditions in NES cells of subjects (II:1 and II:2) and nonrelated controls (C3 and C4). Right: quantification of aminoacylated tRNA Trp of (b) in acidic condition (pH < 5). (c) Representative Western blot analysis of mitochondrial encoded complex IV subunit COI and nuclear encoded complex II subunit A from whole protein extracts of NES cells in nonrelated controls (C3 and C4) and subjects (II:1 and II:2). GAPDH was used as a loading control ( n = 2 independent experiments). (d) Oxygen consumption rates in subjects (II:1 and II:2) and nonrelated control (C3) using glutamate, malate, and pyruvate (GMP + ADP) as electron donors or by uncoupling the membrane potential with CCCP after succinate injection. Data are normalized to protein content in each sample and represented as mean ± standard deviation ( SD ) ( n = 3 independent experiments). (e) Relative enzyme activities of respiratory chain enzyme complex I (NADH coenzyme Q reductase), complex I/III (NADH cytochrome c reductase), complex II (succinate dehydrogenase), complex II/III (succinate:cytochrome c reductase, SCR), and complex IV (cytochrome c oxidase) in isolated mitochondria of subjects (II:1 and II:2) and nonrelated controls (C3 and C4). Data are represented as mean ± standard deviation ( SD ), * p < 0.05, ** p < 0.01 n = 5 independent experiments. (f) Relative enzyme activities of respiratory chain enzyme complex I (NADH‐coenzyme Q reductase) and of CI/III ratio in isolated mitochondria of subject II:2 and C1 control, nucleofected with either pIRES‐eGFP (E.V.) or a pIRES‐eGFP‐WARS2 construct. Data are represented as mean ± standard deviation ( SD ), n = 3 independent experiments. (g) Representative immunostaining analysis with DAPI (blue) and beta‐III tubulin (green) of neurones derived from subject (II:1) and control (C3) NES cells. Scale bars 20 µm.
Article Snippet: Protein extracts were then separated on a 12% NUPAGE acrylamide gel (Invitrogen), transferred to a PVDF membrane (Millipore) and then probed with following antibodies: complex IV subunit COI (Invitrogen 459,600, 1:1,000), complex II subunit A (Abcam ab14715, 1:2,000), and GAPDH (V‐18) (Santa Cruz SC‐20357, 1:1,000).
Techniques: Northern Blot, Control, Western Blot, Membrane, Injection, Standard Deviation, Isolation, Construct, Immunostaining, Derivative Assay
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