459140

antibody from Invitrogen Antibodies

Targeting: UQCRC1 D3S3191, QCR1, UQCR1

Provider product page for 459140

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [45]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [22]

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Product number: 459140 - Provider product page
Provider: Invitrogen Antibodies
Product name: UQCRC1 Monoclonal Antibody (16D10AD9AH5)
Antibody type: Monoclonal
Antigen: Other
Description: Use Human, Cow, Rat or Mouse heart mitochondrial lysate, HepG2 mitochondrial lysate. This antibody gave a positive result when used in the following methanol fixed cell lines: HCT116 for positive control.
Reactivity: Human, Mouse, Rat, Bovine
Host: Mouse
Isotype: IgG
Antibody clone number: 16D10AD9AH5
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C, do not freeze

UQCRC1 protein structure - 459140 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UQCRC1 in heart mitochondria extracts using a UQCRC1 Monoclonal Antibody (Product # 459140) at a concentration of 0.5 µg/mL. Lane 1 : Human heart mitochondrial lysate, Lane 2 : Cow heart mitochondrial lysate, Lane 3 : Rat heart mitochondrial lysate, Lane 4 : Mouse heart mitochondrial lysate, Lane 5 : HepG2 mitochondrial lysate. Predicted band size: 53 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: Western blot was performed using Anti-UQCRC1 Monoclonal Antibody (16D10AD9AH5)(Product # 459140) and a 50 kDa band corresponding to UQCRC1 was observed across all cell lines and tissues tested. Whole Cell Extract-WCL (30 µg lysate) of HeLa (Lane 1), A431 (Lane 2), HepG2 (Lane 3), HEK293 (Lane 4), PC3 (Lane 5), SHSY5Y (Lane 6), Mouse Brain (Lane 7), Mouse Heart (Lane 8) and Mouse Liver (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 ASK1 is involved in brown adipocyte-selective gene expression. ( a ) Oil red O staining of differentiated brown adipocytes (day 6). ( b ) Western blot against indicated genes in brown adipocytes. The same amount of protein was loaded in each lane. ( c ) Western blot against indicated genes in brown adipocytes (day 6). ( d ) Western blot analysis to detect Ucp1 expression in differentiated adipocytes (day 6) in which WT or kinase-negative mutant (KM) ASK1 was overexpressed. ( e ) Intracellular thermometry using fluorescent nanogel thermometer ( n =11 (WT), n =22 (ASK1KO)). Estimated temperature change for each point is shown as an insert. *** P

Supportive validation

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Experimental details: Figure 3 cAMP signalling activates the ASK1-p38 axis and leads to Ucp1 expression. ( a ) ASK1 and p38 activation in response to cAMP signalling in immature brown adipocytes. ( b ) Temporal change of ASK1 and p38 activation status. ( c , d ) Effect of ASK1 deficiency on cAMP signalling-dependent p38 activation. ( e ) Effect of H89 treatment on CL316,243-induced activation of the ASK1-p38 pathway. Cells were pretreated with H89 for 30 min before addition of CL316,243. ( f ) Activation levels of ASK1 in PKIalpha-infected cells. Cells were infected with adenovirus on day -2 and day 0 with 500 MOI. ( g ) Ucp1 and Cidea induction in immature brown adipocytes.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 ASK1 is involved in browning. ( a ) CL316,243 or PBS was injected once a day for 2 days, and iWAT was subjected to qRT-PCR analysis ( n =5). ** P

Supportive validation

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Experimental details: Figure 2 miR-181a inhibits mitochondrial uncoupler-induced mitophagy A. The relative expression of miR-181a in SH-SY5Y cells transfected with miR-181a mimics or the negative control (NC). B. SH-SY5Y cells transfected with miR-181a or NC were treated with 10 muM FCCP or C. CCCP for 12 h. Samples were collected for western blot to analyze the expression of TIM23, COXIV, C-III core, MFN1, LC3, p62, and Actin. Densitometric ratios of mitochondrial marker proteins are quantified by Image J software. D. SH-SY5Y cells transfected with NC or miR-181a were exposed to FCCP (10 muM) and HCQ (20 muM) for 12 h. Western blot was performed to analyze the status of TIM23, LC3 and Actin. E. Image J densitometric analysis of the TIM23/Actin ratios from immunoblots is shown (data from three independent experiments, *p

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 1. IKKalpha regulates mitochondrial biogenesis and fiber type specification in vivo. (A) Hind limbs from four independent litters of IKKalpha +/+ and IKKalpha -/- embryos (total n = 8) were isolated at E20. Cytochrome c and COX Va were measured by real-time quantitative RT-PCR. (B) Additional tissues from E20 limbs from A were homogenized and probed by Western blotting. (C) Total DNA was extracted from E20 limbs, and real-time RT-PCR analysis was performed to quantitate mitochondrial and nuclear (GAPDH) genome copy number. Results are means of five mice per genotype and calculated as fold mitochondrial over genomic DNA. P = 0.004. (D) AAV-GFP and AAV-IKKalpha viruses were injected into the TA. Mice were sacrificed after 3-4 mo, and protein lysates were prepared and analyzed by Western blotting. Shown is a representative blot from five independent experiments. (E and F) After similar viral injections as described in D, mitochondria were fractionated, and protein concentrations or DNA content was determined and normalized to total protein/DNA content (E; n = 5 per group; P < 0.005) or TA muscles were analyzed by EM (F). Bar, 1 um. Parallel muscles were stained for SDH. Bar, 200 um. Arrowheads point to individual mitochondria. (G) TA muscles were isolated from AAV-GFP- and AAV-IKKalpha-injected mice ( n = 3 per group, repeated five times), and myosin isoforms were measured by real-time RT-PCR. The data shown are from a single representative experiment out of three replica

Supportive validation

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Experimental details: Figure 6 Silencing endogenous Parkin inhibits mitophagy A. SH-SY5Y cells were transfected with control or Parkin siRNA for 36 h. Cells were treated with 10 muM FCCP for 12 h. Sample were collected for western blot to analyze the expression of TIM23, MFN1, Parkin, C-III core 1, p62, LC3 and Actin. B. SH-SY5Y cells transfected with control or Parkin siRNA were exposed to FCCP and HCQ for 12 h. Western blot was performed to analyze the status of TIM23, LC3 and Actin. C. Image J densitometric analysis of the TIM23/Actin ratios from immunoblots is shown (data from three independent experiments, *p

Supportive validation

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Experimental details: Fig 7 Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and beta-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean +- SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

Supportive validation

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Experimental details: Fig. 5 Proteome-wide differences in steady-state protein levels between mouse and naked mole rat cells. A , the volcano plot of the p -value versus log 2 ratio of expression levels in naked mole rat and mouse cells. Blue points represent all proteins, and red points highlight proteins involved in glycolysis and oxidative phosphorylation with significantly altered expression levels. B , distribution of log 2 expression level ratios for specified protein subsets. The box plot representations are as described in Figure 1 . Red boxes highlight GO terms involved in ATP production. *, and *** indicate p -values of less than 0.05, 0.005, and 0.0005, respectively, in comparison with the global distribution using the Mann-Whitney U test. C , western blots of selected proteins from respiratory chain complexes (Uqcrc1, Atp512, Cox4) and glycolysis (Eno1, Gapdh). D , measurements of geometric mean intensities of MitoTracker mitochondrial staining of naked mole rat and mouse cells. GO, gene ontology.

Supportive validation

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Experimental details: Fig. 7 Validation of UQCRC1 as an unexpected ClpXP substrate. A , UQCRC1 N termini abundance. MTS (yellow) and mature protein (blue) are shown with starting position of detected N termini. Above and below the protein, identified N-terminal peptides (underlined) are shown with the associated log 2 ( Clpp -/- /wt) and the preceding sequence. Amino acids at the P1 position are highlighted in red. N termini with increased abundance are depicted in green, with decreased abundance in red and unchanged abundance in gray. Predicted masses of the corresponding proteoforms are indicated below the scheme. B , Western blots of UQCRC1 steady state levels in isolated mitochondria. Citrate synthase (CS) was used as loading control. C , CHX chase experiment of UQCRC1 in MEFs. ACTIN was used as loading control ().

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 MTO1 defective cells exhibit proteostasis stress and an altered bioenergetic state. (A and B) Representative immunoblots showing the expression of CLPP, AFG3L2 and LONP1 in extracts of WT and MTO1 HF ( A ), and in MTO1 siRNA 1-, MTO1 siRNA 2- and Negative Control (NC) siRNA-transfected 143B cells ( B ). Porin was used as a loading control. Full-length blots are included in supplementary information (Fig. S 19 ). The scatter plots show the densitometric measurements of the mitoproteases normalized to the loading control and represented as fold change relative to control cells. (C and D) Representative Blue Native-PAGE of OXPHOS complexes in WT and MTO1 HF ( C ), and in MTO1 siRNA 1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells ( D ). Full-length blots and lower-exposure blots of complex III are included in supplementary information (Fig. S 20 ). The scatter plots show the densitometric measurements of OXPHOS complexes normalized to complex-II (loading control) and represented as fold change relative to control cells. (E) Analysis of oxygen consumption rate (OCR) of intact cells using different OXPHOS inhibitors. OCR was measured in each cell type under basal conditions and after sequential addition of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP), rotenone and antimycin A. The scatter plot shows basal OCR (determined as the difference between OCR before oligomycin and OCR after rotenone/antimycin A), ATP-linked OCR (difference b

Supportive validation

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Experimental details: Figure 1 CHCHD 10 resides in the same complex as mitofilin, CHCHD 3 and CHCHD 6 Second dimension of the blue native ( BN )- PAGE showing that CHCHD 10 migrates with the MICOS protein subunits mitofilin, CHCHD 3 and CHCHD 6 in isolated mitochondria from mouse brain. BN - PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P1, P2) fibroblasts. Complexes I to IV of OXPHOS were detected with the 39 kDa subunit antibody ( CI ), 70 kDa subunit antibody ( CII ), core I antibody ( CIII ), and cytochrome c oxidase subunit I antibody ( CIV ). MICOS complex was detected with an anti-mitofilin antibody. Second dimension of the BN - PAGE showing that the steady-state levels of assembled CHCHD 10 in MICOS complex are decreased in patient fibroblasts. The dividing lines correspond to gel sections visible in the raw data file. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN - PAGE from isolated mitochondria permeabilized with 6 g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC , supercomplexes I+ III 2 + IV n . The dividing lines correspond to gels sections visible in the raw data file. Source data are available online for this figure.

Supportive validation

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Experimental details: Figure 6 Phospho-poly-Ub is present in both cytosolic and mitochondrial fractions and leads to mitochondrial parkin translocation. ( A - F ) PC12 cells were treated with 200 muM L-DOPA for 14-17 hours ( A , C , E ) or 10 muM CCCP for 6 hours ( B , D , F ) before being harvested for sub-cellular fractionation. 15% of the resulting cytosolic fractions and 100% of the mitochondria-enriched fractions were analyzed by Western immunoblotting for the indicated proteins. ( A , B ) Representative Western blots showing the distribution of phospho-ubiquitin, parkin, GAPDH, UQCRC1, and Tom20 between cytosolic and mitochondrial fractions. ( A - D ) Phospho-poly-ubiquitin is present both in the cytosol and on mitochondria after L-DOPA ( A , C ) and CCCP ( B , D ) treatment. The phospho-poly-ubiquitin signal in the cytosolic and mitochondrial fractions was normalized to the percentage of the fraction that was loaded to estimate the total level of phospho-poly-ubiquitin in each fraction. The same quantification was carried out for mitochondrial (Tom20, UQCRC1) and cytosolic (parkin, GAPDH) proteins from untreated cells to confirm that fractionation was successful. ( A , B , E , F ) Parkin protein translocates to mitochondria after L-DOPA ( A , E ) and CCCP ( B , F ) treatment. Parkin levels in the mitochondrial fraction with drug or control treatment were normalized to levels of UQCRC1 and plotted in ( E , F ). ( C - F ) Error bars show SEM from N = 4 ( C , D ), 7 ( E ), and 4 ( F ) independ

Supportive validation

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Experimental details: Figure 9 Lack of robust mitophagy in the CCCP and L-DOPA models. ( A , B ) L-DOPA and CCCP do not induce changes in mitochondrial protein levels consistent with robust mitophagy. Differentiated PC12 cells were treated with 10 muM CCCP ( A ) or 200 muM L-DOPA ( B ) for the indicated times before harvest for WB. Representative blots and quantifications of the indicated proteins from N = 3 independent experiments are shown. ( A ) CCCP does not significantly decrease levels of UQCRC1, VDAC, or Tom20, while it does induce loss of parkin, Mfn2, Tim23, and Opa1-long. ( B ) L-DOPA does not significantly decrease levels of Mfn2, UQCRC1, VDAC, Tim23, or Tom20, while it does induce loss of parkin and Opa1-long. Error bars show SEM; *p

Supportive validation

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Experimental details: Figure 6 CsA prevents Mn-induced Opa-1 cleavage and Drp-1 translocation to mitochondria. Opa-1 expression levels in total cell lysates. Anti-beta- Actin antibody was used as a loading control ( A ); Drp-1 expression levels in enriched-mitochondrial and cytosolic fractions. Membranes were stripped and reprobed for anti-Complex III subunit core 1- OxPhos (Complex III) I and anti-beta- Actin as loading control ( B ); CsA diminishes Mn-induced mitochondrial fragmentation, loss of Deltapsim and apoptotic nuclei appeareance. Quantification of mitochondria ( C ) and nuclei morphology ( D ) classified according to Figs. 4, 5. Scale bar: 10 um. Three independent experiments were conducted. Statistical significance, **p