Anti-NDUFB8 抗体 [20E9DH10C12]

• WB

Unknown

Western blot - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.

All lanes:

Western blot - Anti-NDUFB8 antibody [20E9DH10C12] (ab110242) at 0.5 µg/mL

Lane 1:

Isolated mitochondria from Human heart at 5 µg

Lane 2:

Isolated mitochondria from cow heart at 1 µg

Lane 3:

Isolated mitochondria from rat heart at 10 µg

Lane 4:

Isolated mitochondria from mouse heart at 10 µg

Predicted band size: 22 kDa

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• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Skeletal muscle immunohistochemistry using ab110242 on frozen tissue sections from a patient with a single large deletion of the mtDNA show a mosaic of complex I positive and complex I negative fibers.

• IHC-Fr

PubMed

Immunohistochemistry (Frozen sections) - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Immunohistochemistry in serial sections of the muscle of patient AIV-5

The patient is sufferening from Spastic paraplegia 7.

Immunohistochemistry for complex I (NDUFB8) (A), complex II (B), complex III (C) and COX/SDH histochemistry (D) in serial sections of the muscle of patient AIV-5. There are complex I, III and IV deficient fibres, but complex I deficiency is most pronounced. Arrows mark serial sections of the same muscle fibers stained for different complexes.

NDUFB8 (also refered to as complex I) was detected using ab110242 at 1/100 dilution).

(After Figure 3 of Wedding et al)

Wedding, I.M. et al PLoS One. 2014 Jan 22;9(1):e86340. doi: 10.1371/journal.pone.0086340. eCollection 2014. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

CiteAb

Western blot - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Western Blotting using Anti-NDUFB8 antibody [20E9DH10C12], ab110242. Publication image from Protasoni, M. et al., 2020, EMBO J, 31912925. Legend direct from paper.

Reduced steady‐state levels of structural MRC subunits in δ4‐CYB cellsScatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy‐to‐light (H/L) and light‐to‐heavy (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and δ4‐CYB cells (see also Fig EV1). The logarithmic ratios were calculated using MaxQuant (Cox & Mann, 2008), and the statistical significance of the differences for the enrichment or depletion of the proteins was determined with Perseus (Cox & Mann, 2011; Tyanova et al, 2016).Labeling of the thirteen mtDNA‐encoded MRC structural subunits. Cells were incubated with [35S]‐L‐Met for 1 h in the presence of emetine 100 µg/ml to inhibit cytoplasmic translation.Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; ***P = 0.0007.Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001; **P = 0.0024 (NDUFS3); **P = 0.0061 (NDUFB8).Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐Actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test **P = 0.0011.Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). Two‐way ANOVA with Sidak's multiple comparisons test ****P < 0.0001.Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS–PAGE, from three independent replicates of WT and δ4‐CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β‐actin. The mean of the three control (WT) samples was set to 1.0, and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n = 3). There were no differences in the steady‐state levels of the tested subunits (2‐way ANOVA with Sidak's multiple comparisons test).Source data are available online for this figure.

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• WB

CiteAb

Western blot - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Western Blotting using Anti-NDUFB8 antibody [20E9DH10C12], ab110242. Publication image from Carbognin, E. et al., 2016, EMBO J, 26903601. Legend direct from paper.

LIF/Stat3 activates mitochondrial respirationOxygen consumption rate (OCR) measured by Seahorse extracellular flux assay of Stat3+/+ and Stat3−/− cells maintained in 2i condition in the presence of LIF; 200 nM FCCP (a mitochondria uncoupler) treatment resulted in higher OCR increase in Stat3+/+ compared to Stat3−/− cells, showing a higher level of maximal mitochondrial electron transport chain (ETC) activity in Stat3+/+ cells. Injection of 200 nM antimycin shows similar non‐mitochondrial respiration rates for both Stat3+/+ and Stat3−/− cells. Mean and s.e.m. of 5 technical replicates are shown.Oxygen consumption rate (OCR) of Stat3+/+ cells cultured in 2i conditions without LIF or with LIF for several passages; 200 nM FCCP and 200 nM antimycin were injected and resulted in a higher mitochondrial respiration activity in cells cultured in the presence of LIF. Mean and s.e.m. of 4 replicates are shown. See also Appendix Fig S3D.Relative changes in oxygen consumption after 200 nM FCCP treatment of Stat3+/+ cells cultured in 2i media in the presence (dark blue bars) and absence of LIF (light blue bars). Mean and s.e.m. of > 4 technical replicates of three independent experiments are shown. Unpaired t‐test : *P < 0.05.Western blot of Stat3+/+ cells cultured in the presence or absence of LIF. Note that protein levels of two mitochondrial markers (TOM20 and TIMM23) do not change in the absence of LIF. GAPDH was used as a loading control. Relative mean intensity is shown below each band.Mitochondrial DNA expression analysis of Stat3+/+ cells maintained in 2i in the presence (dark blue bars) or absence (light blue bars) of LIF. The abundance of 3 mitochondrial genomic loci was measured and normalized to a nuclear genomic locus on chromosome 3. Mean and s.e.m. of three independent biological replicates are shown.BNGE analysis followed by Western blot for a Complex I protein (NDUFB8). ATPase serves as a loading control. RCS, respiratory chain supercomplexes. See also Appendix Fig S3E and F.Quantification of the chemiluminescent RCS/ATPase signal ratio (left) and Complex I/ATPase signal ratio (right). Mean and s.d. of three independent experiments. Unpaired t‐test : *P < 0.05, **P < 0.01, ***P < 0.001.Source data are available online for this figure.

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• WB

CiteAb

Western blot - Anti-NDUFB8 antibody [20E9DH10C12] (AB110242)

Western Blotting using Anti-NDUFB8 antibody [20E9DH10C12], ab110242. Publication image from Kopajtich, R. et al., 2017, Nat Commun, 28604674. Legend direct from paper.

RNA aberrant expression detection and validation.(a) Aberrantly expressed genes (Hochberg corrected P value<0.05 and |Z-score|>3) for each patient fibroblasts. (b) Gene-wise RNA expression volcano plot of nominal P values (−log10P value) against Z-scores of the patient #35791 compared against all other fibroblasts. Z-scores with absolute value >5 are plotted at ±5, respectively. (c) Same as (b) for patient #73804. (d) Sample-wise RNA expression is ranked for the genes TIMMDC1 (top) and MGST1 (bottom). Samples with aberrant expression for the corresponding gene are highlighted in red (#35791, #66744, and #73804). (e) Gene-wise comparison of RNA and protein fold changes of patient #35791 compared to the average across the fibroblast cell lines of all other patients. Subunits of the mitochondrial respiratory chain complex I are highlighted (red squares). Reliably detected proteins that were not detected in this sample are shown separately with their corresponding RNA fold changes (points below solid horizontal line). (f) Western blot of TIMMDC1, NDUFA13, NDUFB3 and NDUFB8 protein in three fibroblast cell lines without (#62346, #91324, NHDF) and three with a variant in TIMMDC1 (#35791, #66744 and #96687), and fibroblasts re-expressing TIMMDC1 (‘-T’) (#35791-T, #66744-T and #96687-T). UQCRC2 was used as loading control. CI, complex I subunit; CIII, complex III subunit; MW, molecular weight. (g) Blue native PAGE blot of the control fibroblasts re-expressing TIMMDC1 (NHDF-T), the control fibroblasts (NHDF), patient fibroblasts (#96687) and patient fibroblast re-expressing TIMMDC1 (#96687-T). Immunodecoration for complex I and complex III was performed using NDUFB8 and UQCRC2 antibodies, respectively. CI, complex I subunit; CIII, complex III subunit.

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