459210

antibody from Invitrogen Antibodies

Targeting: NDUFB8 ASHI, CI-ASHI

Provider product page for 459210

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Western blot [3]
Immunohistochemistry [1]
Other assay [16]

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Product number: 459210 - Provider product page
Provider: Invitrogen Antibodies
Product name: NDUFB8 Monoclonal Antibody (20E9DH10C12)
Antibody type: Monoclonal
Antigen: Other
Description: Positive control: Isolated mitochondria from Human heart, bovine heart, rat heart and mouse heart. Skeletal muscle tissue.
Antibody clone number: 20E9DH10C12
Concentration: 1 mg/mL

NDUFB8 protein structure - 459210 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NDUFB8 in isolated mitochondria using an NDUFB8 Monoclonal antibody (Product # 459210) at a concentration of 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. 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NDUFB8 was achieved by transfecting HepG2 with NDUFB8 specific siRNAs (Silencer® select Product # s9368, s9369 ). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the NDUFB8 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NDUFB8 Monoclonal Antibody (20E9DH10C12) (Product # 45-9210, 1 µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to NDUFB8.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NDUFB8 Monoclonal Antibody (20E9DH10C12) (Product # 45-9210) and a 18 kDa band corresponding to NDUFB8 was observed across all the cell lines and tissues tested along with a 25 kDa band in the Mouse Barin, Mouse Heart and Mouse Liver. Membrane enriched extracts (60 µg lysate) of A-431 (Lane 1), HEK-293 (Lane 2), Hep G2 (Lane 3), tissue extracts of Mouse Brain (Lane 4), Mouse Heart (Lane 5), Rat Heart (Lane 6), Mouse Skeletal Muscle (Lane 7) and Mouse Liver (Lane 8) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/ml) and detected by chemiluminescence with Goat Anti-Mouse IgG Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 The mitochondrial translation machinery regulates global mitochondrial activity and Emu -myc cell growth A. Emu- myc lymphoma cells were transduced with shRNAs against Ptcd3 , Mrps5 , Mrps27 , Renilla luciferase (Ren) or a control empty vector (EV). The number of viable cells at each time point (top) was determined by trypan blue staining. For each single shRNA the efficiency of knockdown was evaluated by real-time qPCR three days post-transduction (bottom). Transcript abundance is expressed as mean +- s.d. of triplicate measurements, expressed relative to the EV control and normalized to Rplp0 . B. Doubling time was calculated from the growth curves in A., using the formula described in experimental procedures. C. Cell death was evaluated by Annexin V and propidium iodide (PI) staining at the 48hrs time-point. The graph shows the percentage of Annexin V+/PI- (black) and Annexin V+/PI+ cells (grey) corresponding to early and late apoptotic cells, respectively. D. Western blot analysis of components of the Electron Transport Chain (ETC) Complexes I-IV following 48hrs of knock-down. As loading controls, we used antibodies against cytoplasmic Vinculin and the mitochondrial Voltage-dependent anion channel (VDAC). E. Real-time qPCR quantification of mtDNA, normalized to nuclear DNA. Results are shown as mean +- s.d. from triplicate measurements. F. Oxygen consumption rate (OCR) was determined as described in experimental procedures at 48hrs. Data were normalized to total

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Tigecycline impairs the growth of Emu -myc lymphoma cells and inhibits translation of mitochondrial proteins A. Dose-response curve of Emu- myc lymphoma cells treated with the indicated doses of Tigecycline (Tig), Linezolid (Lin) or Cloramphenicol (Cam) for 48hrs. Data are shown as cell numbers (left panel) or viability (right panel), as percentage of untreated cells (mock-treated with DMSO). Cell number and viability was determined by trypan blue staining. B. Oxygen consumption rate (OCR) measured as defined in Figure 2F on Emu- myc lymphoma cells treated with the indicated antibiotics or carrier (DMSO). To measure spare respiratory capacity, cells were treated with the ETC uncoupler 2,4-Dinitrophenol (DNP). C. Western blot analysis of components of the ETC complexes I-IV. Cells were treated with the indicated concentration of Tigecycline for 24 or 48hrs. The loading controls are Vinculin and VDAC, as in Figure 2D . D. Mitochondrial membrane potential was determined by staining with the cationic cyanine dye DilC1(5). E. Cleaved caspase 3/7 activity was determined with the caspase 3/7 glo assay luminescent kit. Measurements in D and E were taken after 6 hrs of treatment. Means, s.d. and statistical significance are as defined in Figure 2 . See also Supplementary Figures S3 and S4 .

Supportive validation

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Experimental details: Fig 5 Preserved OXPHOS protein levels and mitochondrial biogenic signaling in ADQ -/- hearts. AMPK phosphorylation (A), SIRT1 activity (B), lysine acetylation (Lys-Ac) of PGC-1alpha (C), mRNA expression of OXPHOS subunits (D), mRNA expression of mitochondrial biogenesis signaling molecules (E), mRNA expression of fatty acid oxidation genes and PPARalpha (F), and mitochondrial protein levels of OXPHOS complexes I (NDUFB8 subunit; G), II (Fp subunit; H), and IV (subunit IV; I) in hearts of ADQ -/- and WT mice at 8 weeks of age; n = 4-5. Myocardial mRNA expression is expressed relative to WT expression which was set to 1 (indicated by the dotted line). * p

Supportive validation

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Experimental details: Fig. 5 AIF selectively controls respiratory chain protein expression in pancreatic cancer cells. Following suppression of AIF, respiratory chain status was assessed by immunoblot analysis of complex I (39-, 20-, and 17-kDa subunits) and COX IV

Supportive validation

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Experimental details: Figure 1 Exercise training increase mitochondrial oxidative phosphorylation complexes in an AMPK alpha2-dependent manner . Protein levels of oxidative phosphorylation complexes, (A) Complex I, (B) Complex II, (C) Complex III, (D) Complex IV, (E) Complex V (oligomycin-sensitivity conferring protein subunit, OSCP), and (F) Cytochrome C were evaluated in quadriceps muscle of sedentary (control) or exercise trained female WT and AMPK alpha2 KD mice ( n = 13-15). Values are mean +- SEM. * indicates vs. WT control ( p < 0.05) analyzed by unpaired t -tests.

Supportive validation

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Experimental details: Figure 4 Repeated treatment with AICAR increases abundance of mitochondrial electron transport chain proteins in an AMPK alpha2-dependent manner . WT and AMPK alpha2 KD male mice were given daily subcutaneous injections with AICAR (500 mg/kg body weight) or saline for 4 weeks. (A-E) Protein abundance of oxidative phosphorylation Complexes I-V and (F) Cytochrome C was measured in quadriceps muscle ( n = 7-8). A significant interaction effect (treatment x genotype; p < 0.05) was observed for Cytochrome C. Values are mean +- SEM. * indicates vs. WT saline ( p < 0.05) analyzed by t -tests, ** indicates vs. WT saline ( p < 0.01), and ++ indicates genotype effect within AICAR treated animals ( p < 0.01).

Supportive validation

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Experimental details: Figure 3 DGUOK regulates NAD + biosynthesis through NMNAT 2 . (A) Western blot analysis of NMNAT2 and NDUFB8 expression level in NMNAT2 -KD and control H1650 cells. (B) LC-MS analysis of NAD + level in NMNAT2 -KD and control H1650 cells. (C) Quantification of NAD + level in NMNAT2 -KD and control H1650 cells. (D) Western blot analysis of NMNAT2, NDUFB8 and DGUOK expression levels in DGUOK -KO, DGUOK -KO/ NMNAT2 -OE and control H1650 cells. (E) LC-MS analysis of NAD + level in DGUOK -KO, DGUOK -KO/ NMNAT2 -OE and control H1650 cells. (F) Quantification of NAD + level in DGUOK -KO, DGUOK -KO/ NMNAT2 -OE and control H1650 cells. ***p < 0001.

Supportive validation

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Experimental details: Figure 4 DGUOK regulates NMNAT2 independent of mitochondria complex I activity. (A) Western blot analysis of DGUOK in control and Rotenone treated H1650 cells. (B) qRT-PCR analysis of the NMNAT2 mRNA level in Rotenone treated and control H1650 cells. (C) Western blot analysis of NDUFB8 in NDUFB8 -KD and control H1650 cells. (D) qRT-PCR analysis of the NMNAT2 mRNA level in NDUFB8 -KD and control H1650 cells. (E) BN-PAGE gel analysis of mitochondria complex I level in NMNAT2 -KD and control H1650 cells. n.s. not significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Tau oligomers induce mitochondrial alterations . (A) Representative western blot of mouse brain homogenate. The levels of complex I, complex V, and caspase-9 were measured by band quantification and normalized with the levels of tubulin. Abbreviations are as in Fig. 4. (B) Complex I levels were significantly lower in the hemisphere injected with tau oligomers in comparison with the ones injected with fibrils or monomers. (C) No differences in the levels of complex V were observed in any of the groups. (D) Caspase-9 activation was significantly higher in tau oligomer groups over both tau fibril- and monomer-injected groups. (E-G) Double staining between human tau-specific antibody HT7 (green fluorescence) and mitochondrial porin antibody (red fluorescence) demonstrates the internalization of tau oligomers in CA1 cells and their interaction with the mitochondria. (H) The co-localization of tau oligomers with mitochondria was confirmed by ICA of the signal. Data are represented as mean +- SE. *p < 0.01, n = 6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 DGUOK is required for mitochondrial OXPHOS in lung adenocarcinoma cells A Waterfall plot showing fold changes of 17 mitochondrial proteins differentially regulated in DGUOK KO H1650 cells. Fold changes (log 2 ) are presented as mean +- SEM of results from two independent experiments. B Top enriched pathways in DGUOK high lung adenocarcinoma ( n = 100) compared to DGUOK low lung adenocarcinoma ( n = 100) based on Gene Set Enrichment Analysis on RNA-sequencing data from the TCGA database. NES, normalized enrichment score, Norm. p , normalized P value. C Enrichment score profile of the oxidative_phosphorylation pathway in GSEA in (B). D Western blotting showing the decrease in respiration complex I subunits in DGUOK KO H1650 cells. E The effects of DGUOK depletion on mitochondrial oxygen consumption rate in H1650 cells. n = 3 independent replicates per group. F The effects of DGUOK KO on mtDNA copies in H1650 cells. Two pairs of primers recognizing minor Arc (minArc) and major Arc (majArc) region of mtDNA were used for the qPCR quantitation. G The effect of DGUOK depletion on the activities of respiratory complexes I, II, III, and IV. H Representative IHC staining showing the expression of DGUOK and NDUFB8 in lung adenocarcinoma patient specimens. I Correlation between DGUOK staining intensity and NDUFB8 staining intensity in a cohort of 113 lung adenocarcinoma patients. r s = 0.728, P < 0.0001, as determined by Spearman's correlation test. Data information: Data are sh

Supportive validation

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Experimental details: Figure 7 Genetic targeting of DGUOK inhibits tumor initiation and induces tumor regression A The effect of DDI treatment on mtDNA copies in H1650 cells and three patient-derived cancer cells from lung adenocarcinoma (PDC027, PDC236, and PDC251). Two pairs of primers recognizing minor Arc (minArc) and major Arc (majArc) region of mtDNA were used for the qPCR quantitation. Data are shown as mean +- SD ( n = 3 per group). B The effect of DDI treatment on DGUOK protein levels in the three patient-derived cancer cell lines. C, D Representative images (C) and quantitation data (D) showing the effects of DDI treatment on lung sphere formation in H1650 and patient-derived lung cancer cells. Data are shown as mean +- SD ( n = 4 independent samples per group). E Western blotting showing the doxycycline (doxy)-induced expression of Cas9 and the deletion of DGUOK protein expression after 7 days of treatment. F 1 x 10 5 LLC cells expressing Tet-On Cas9 and DGUOK sgRNA5 (KO2) were inoculated via s.c. into both flanks of Albino BL6 mice. One group of mice ( n = 5 mice, 10 inoculations per group) were provided with doxycycline chow 2 days after inoculation. The formation of tumor was monitored by bioluminescence imaging 28 days postinoculation. P < 0.0001, n = 10 mice per group, Fisher's exact test. G 1 x 10 5 LLC cells stably expressing Tet-On Cas9 and with DGUOK sgRNA (KO2 group) or without sgRNA (control group) were inoculated via s.c. into Albino BL6 mice. Mice were provided with doxycyc