Fig 1: Identification of Complex I FMN and FAD of aKGDHC in DDM-solubilized brain and heart mitochondria. (A) Intact mitochondria were solubilized using a DDM/protein ratio of 3.6 g/g. Identical aliquots of 15 µg of total mitochondrial protein were loaded on an acrylamide gradient gel (3–12%). Heart (right) and brain (left) mitochondria samples were applied on the same gel and hrCN electrophoresis was performed as in Ref. [38]. Lanes 2, Coomassie stained gel strips showing the presence of complexes (I–V) with molecular weight markers (Lane 1); Lanes 3, complex I in gel activity strips; Lanes 4 and 5, flavin fluorescence from the same strip before (4) and after (5) treatment with 20% SDS (Excitation/Emission = 473/530 nm). Lanes 6, complex I immunodetection by Western blot using antibody against NDUFS8 subunit; (B) dependence of the flavin fluorescence intensity in the complex I (black circles) and aKGDHC (grey circles) bands upon protein load on the gel. Values are mean ± SEM, n = 4; (C) calibration curve for flavin fluorescence signal made of standard free FMN solution applied directly on a gel and intensity of fluorescent spots measured (mean ± SEM, n = 3).
Fig 2: Intraperitoneal administration of NaB protects mitochondria via anti-oxidative stress at 24 h after ICH. (A) Representative Western blot images. Quantitative analyses of (B) NDUFS8 and (C) DJ-1 levels in mitochondria. (D) Level of ATP at 24 h after ICH; (E) Level of ROS at 24 h after ICH. n = 6 for each group. The bars represent the mean ± SD. *p < 0.05 vs. sham, #p < 0.05 vs. ICH + vehicle, &p < 0.05 vs. ICH + NaB.
Fig 3: Specific features of an hrCN gel using DDM-solubilized intact brain mitochondria.A, mouse brain mitochondria were solubilized using a DDM/protein ratio of 3.6 g/g. Identical aliquots (15 µg) were loaded on a polyacrylamide gradient gel (3–12%). hrCNE was performed as described (76). Lane 2, Coomassie-stained gel strips showing the presence of complexes (I–V). Lane 1, the position of fluorescently labeled molecular weight markers in kilodalton is shown to the left; lane 3, complex I in-gel activity strip; lane 4, complex I immunodetection by Western blot with antibody against NDUFS8 subunit; lanes 5 and 6, flavin fluorescence signal from the same strip before (5) and after (6) 20 min incubation with 20% SDS, respectively. Mass spectrometric protein identifications for flavin-containing bands F1 and F2 from an untreated hrCN gel are provided in Table 1. B, averaged fluorescence emission spectrum (excitation at 450 nm) of the extract of flavin-containing band F2 (mean ± SEM, n = 3). DDM, n-dodecyl-ß-d-maltoside.
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