459100

antibody from Invitrogen Antibodies

Targeting: NDUFA9 CI-39k, COQ11, NDUFS2L, SDR22E1

Provider product page for 459100

Western blot

Other assay

Antibody data

Antibody Data
Antigen structure
References [57]
Comments [0]
Validations
Western blot [2]
Other assay [22]

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Product number: 459100 - Provider product page
Provider: Invitrogen Antibodies
Product name: NDUFA9 Monoclonal Antibody (20C11B11B11)
Antibody type: Monoclonal
Antigen: Other
Description: Purification is near homogeneity as judged by SDS-PAGE. The antibody was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation.
Antibody clone number: 20C11B11B11
Concentration: 1 mg/mL

NDUFA9 protein structure - 459100 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of NDUFA9 was achieved by transfecting Hep G2 cells with NDUFA9 specific siRNAs (Silencer® select Product # s39346, s9347). Western blot analysis (Fig. a) was performed using membrane enriched cell extracts from the NDUFA9 KD cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NDUFA9 Monoclonal Antibody (20C11B11B11) (Product # 45-9100, 1µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 0.25 µg/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 NDUFA9.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NDUFA9 Monoclonal Antibody (20C11B11B11) (Product # 45-9100) and a 36 kDa band corresponding to NDUFA9 was observed across cell lines and tissues tested. Membrane enriched cell extracts (30 µg lysate) of HeLa (Lane 1), Caco-2 (Lane 2), SW480 (Lane 3), Hep G2 (Lane 4), Jurkat (Lane 5), K-562 (Lane 6), MCF-7 (Lane 7), Mouse Liver (Lane 8), Rat Liver (Lane 9), Mouse Brain (Lane 10) and Rat Brain (Lane 11) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # ) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence using 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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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 Characterization of TFAM and Twinkle (TW) mice. (A) Expression of human TFAM (hTFAM) in left ventricle (LV) and aorta in TFAM and wild-type (WT) control mice. (B) mtDNA copy number in myocardium from TFAM and TW mice by real-time PCR (n = 4). (C) Expression of endogenous murine TFAM (mTFAM) and mitochondrial complex proteins in LV of TFAM and TW mice. (D) Transcription of mtDNA-encoded genes in TFAM and TW mice (n = 6). Data are expressed as mean +- SEM. * P < 0.05 vs. WT, ** P < 0.01 vs. WT, analyzed by Student's t- test.

Supportive validation

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Experimental details: Figure 5. Liver-specific Cluh deletion affects mitochondrial distribution and structure, assembled respiratory supercomplexes, and respiratory capacity. (A) Representative confocal images of livers of 8-wk-old mice of the indicated genotypes. To analyze mitochondrial morphology, mice were crossed with a stop-mito-YFP reporter line activated by Cre recombination. n = 4. Bars, 5 um. (B) Oxygen consumption of mitochondria isolated from livers of 8-wk-old mice. State III respiration was measured in the presence of pyruvate, malate, glutamate, and ADP (complex I [CI]), followed by addition of succinate (complex I + complex II [CI + CII]). The proton leak was measured after addition of oligomycin, whereas maximal respiration was assessed by CCCP titration. n = 5. Graph shows means +- SEM. **, P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Cluh -deficient MEFs mimic liver phenotypes. (A) Oxygen consumption of intact MEFs cultured in glucose or galactose medium. The proton leak was measured after the addition of oligomycin, whereas maximal respiration was assessed by CCCP titration. n >= 4. (B) Mitochondrial morphology in MEFs transfected with mito-mCherry. Graphs show the mean aspect ratio (area/perimeter) on the x axis and the number of mitochondria on the y axis for individual cells from three independent experiments. Right panels show representative images of mitochondrial morphology at the indicated time points. Bar, 12 um. (C) Growth curves of MEFs cultured in glucose or galactose medium during five consecutive days. n = 3. (D) Representative images of LD staining in MEFs grown in glucose medium. Nuclei were stained with DAPI (blue), and LDs were stained with BODIPY 493/503 (green). Bar, 20 um. (E) Quantification of LD staining shown in D. 50 cells were analyzed per genotype per experiment. Graph shows the mean area of LDs per cell. n = 3. (A, C, and E) Error bars are means +- SEM. *, 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. COX6B2 enhances complex IV activity of OXPHOS without increasing ROS production. ( A ) Schematic of complex IV activity measurements in isolated mitochondria. Details are described in Material and methods. antA, Antimycin A; Cyt c, Cytochrome c; ATP Syn, ATP synthase. ( B ) Complex IV activity in indicated cell lines using TMPD/ascorbate as a substrate. Bars represent mean + SEM (n >= 8). p-Value calculated by Student's t -test. ( C ) As in ( B ) in HCC515 cell lines expressing CTRL and COX6B1-V5 cDNA. Bars represent mean + SEM (n = 8). p-Value calculated by Student's t -test. ( D ) Indicated lysates from HCC515 cells were run on a BN-PAGE gel and stained with Colloidal Blue (left two panels) or immunoblotted with anti-COX6B2, anti-COX6B1 and anti-COXIV (right panels). Representative image of n >= 3. MW markers are indicated to identify different complexes (). ( E ) Left: Distribution of COX6B1 and COX6B2 incorporated in complex IV monomers, dimers or supercomplexes as detected by BN-PAGE in ( D ). Right: Bars represent mean +- SEM (n >= 3) based on quantification of bands in ( D ). In monomeric complex IV, p value calculated by Mann-Whitney test whereas others calculated by Student's t -test. ( F ) Left: Distribution of COX6B1/COX6B2 incorporated in monomeric complex IV, dimeric complex IV and supercomplexes based on BN-PAGE in COX6B2-V5 or COX6B1-V5 overexpressing cell lines ( D ). Right: Bars represent mean +- SEM (n >= 3) based on quantification of bands in ( D

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Depletion of p150 glued does not induce damaged mitochondria accumulation. (A) Control siRNA or DCTN1 siRNA transfected cells were fixed and co-stained with antibodies against LAMP2 (green) and TOM20 (red), and analyzed using confocal microscopy. Bars, 10 mum. (B) Control siRNA or DCTN1 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies against complex I, TOM20, and actin. (C, D) Densitometry analysis of complex I (C) and TOM20 (D) levels relative to actin was performed. (E, F) DCTN1 siRNA transfected HeLa cells were incubated with Mitotracker-Red CMXRos and intracellular fluorescence intensity was measured by flow cytometry. The histograms of MitoTracker-Red CMXRos fluorescence (E) and the percentages of cells with reduced mitochondrial potentials (F) are shown. The error bar indicates each standard deviation. Statistics are from three independent experiments: N.S., not significant.

Supportive validation

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Experimental details: 5 Figure Loss of TMBIM5 promotes proteolysis by AFG3L2 A Pie chart showing the localization of proteins accumulating at decreased levels in TMBIM5 -/- HeLa cells. Localization according to MitoCarta 3.0. OM, outer membrane; IMS, intermembrane space; IM, inner membrane. B Alterations in mitochondrial proteome in WT and TMBIM5 -/- HeLa cells upon depletion of AFG3L2. WT and TMBIM5 -/- HeLa cells were transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Proteins, which were downregulated in the absence of TMBIM5 and whose steady-state levels were altered upon depletion of AFG3L2, were identified using a two-way ANOVA. An interaction P -value of 0.01 was used as a cutoff. Log2-transformed LFQ intensities of MitoCarta 3.0 proteins were Z -Score normalized and visualized in hierarchical cluster analysis (Euclidean distance, complete method). The dendrogram is omitted. Proteins accumulating upon depletion of AFG3L2 (cluster 1, C1) and proteins whose steady-state levels are decreased upon depletion of AFG3L2 (cluster 2, C2) are shown. See also Dataset EV6. C Representative immunoblot of TMBIM5 -/- HeLa cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide (CHX; 10 mug/ml) and collected at the indicated time points ( n = 3 independent experiments). Data information: See also Dataset EV6. Source data are available online for this figure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Loss of merlin expression in human Schwann cells leads to decreased mitochondrial oxidative phosphorylation complex levels and activity. A, the mitochondrial content of human WT- and MD-Schwann cells was determined after 30 min incubation with the mitochondrial membrane potential-independent probe MitoTracker Green (200 n m ) and expressed as percentage of WT-Schwann cells ( n = 3 with 4 replicates). B and C, representative IR Western blots of mitochondrial proteins in human WT- and MD-Schwann cells: B, Hsp60, VDAC, PD, and cytochrome c (cyt c ); and C, complex I (NDUFA9 subunit), complex II (SDHA subunit), and complex IV (subunit COX IV). Below , quantitation of the corresponding bands normalized against alpha-tubulin and expressed relative to WT-Schwann cells ( n = 4-12). D, mitochondrial complex I, II + III, and IV activities were assessed in disrupted isolated mitochondria of WT- and MD-Schwann cells. Complex I activity was measured at 340 nm by the rotenone-sensitive reduction of ubiquinone-1 in the presence of potassium cyanide and NADH. Complex II + III activity was determined by the antimycin A-sensitive reduction of cytochrome c at 550 nm in the presence of potassium cyanide and succinate. Complex IV activity was determined by monitoring the potassium cyanide-sensitive oxidation of cytochrome c at 550 nm. Activities are expressed as nanomole/min/mg of protein; columns represent the mean +- S.D. ( n = 6-7). E, representative images