MCU - Antibodies - The Human Protein Atlas


	Antibody HPA016480

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich
Product name	HPA016480
Host species	Rabbit
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb
Concentration	0.016 mg/ml
Purity	Affinity purified using the PrEST-antigen as affinity ligand
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	4.1
Referencesⁱ References to publications in which the antibody has been used.	34
Proper citation	Atlas Antibodies Cat#HPA016480, RRID:AB_2071893
Validation summaryⁱ All assays through which the antibody has been validated. Assays&annotation provide a detailed description of the different assays. The pie-charts indicate degree of validation.	ICC IHC WB PA
IMMUNOCYTOCHEMISTRYⁱ Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
Validationⁱ Results of validation by standard or enhanced validation. Standard validation is based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. Standard validation results in scores Supported, Approved or Uncertain. Enhanced validation is performed using either siRNA knockdown, tagged GFP cell lines or independent antibodies. For the siRNA validation the decrease in antibody-based staining intensity upon target protein downregulation is evaluated. For the GFP validation the signal overlap between the antibody staining and the GFP-tagged protein is evaluated. For the independent antibodies validation the evaluation is based on comparison of the staining of two (or more) independent antibodies directed towards independent epitopes on the protein. For all cases except the siRNA validation, an image representative of the antibody staining pattern is shown. For the siRNA validation, a box plot of the results is shown.	Supportedⁱ Reliability scores for antibodies used in immunocytochemistry are set by comparing the staining pattern in cell lines with external experimental evidence for protein localization. The scores are termed Supported, Approved and Uncertain. The subcellular location is supported by literature. Immunofluorescent staining of human cell line U2OS shows localization to mitochondria.

Antibody dilution	Human assay: A-431 fixed with PFA, dilution: 1:8 Human assay: U-251MG fixed with PFA, dilution: 1:8 Human assay: U2OS fixed with PFA, dilution: 1:8
IMMUNOHISTOCHEMISTRYⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain.
Validationⁱ Results of validation by standard or enhanced validation based on assessment of antibody performance in 44 normal tissues. Standard validation results in scores Supported, Approved or Uncertain. An image representative of the antibody staining pattern is shown. Enhanced validation results in the score Enhanced and includes two methods: Orthogonal validation and Independent antibody validation. For orthogonal validation, representative images of high and low expression are shown. For independent antibody validation, four images of each independent antibody are displayed.	Approvedⁱ Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain. Immunohistochemical staining of human duodenum shows strong cytoplasmic positivity in glandular cells. Duodenum

Retrievalⁱ Antigen retrieval is a method used to restore/retrieve the epitope (antibody bidning region) of the target protein, cross-linked, and thus masked, during tissue preserving fixative treatment of the tissues.	HIER pH6
Antibody dilution	1:7
Literature conformityⁱ Conformance of the expression pattern with available gene/protein characterization data in scientific literature and data from bioinformatic predictions. UniProt is used as the main source of gene/protein characterization data and when relevant, available publications and other sources of information are researched in depth. Extensive or sufficient gene/protein data requires that there is evidence of existence on a protein level and that a substantial quantity of published experimental data is available from literature and public databases. Limited protein/gene characterization data does not require evidence of existence on a protein level and refers to genes for which only bioinformatic predictions and scarce published experimental data is available.	Partly consistent with extensive gene/protein characterization data.
RNA consistencyⁱ Consistency between immunohistochemistry data and consensus RNA levels is divided into five different categories: i) High consistency, ii) Medium consistency, iii) Low consistency, iv) Very low consistency, and v) Cannot be evaluated.	Low consistency between antibody staining and RNA expression data.
WESTERN BLOTⁱ A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with unreliable result a revalidation using an over-expression lysate is performed.
Validationⁱ Western Blot is used for quality control of the polyclonal antibodies generated in the project. After purification, the antibodies are used to detect bands in a setup of lysate and different tissues. The result is then scored Enhanced, Supported, Approved, or Uncertain. Enhanced validation includes five different methods: Genetic validation, Recombinant expression validation, Independent antibody validation, Orthogonal validation and Capture MS validation.	Enhanced - Recombinant expressionⁱ This method is based on over-expression of the target protein in a cell line preferably not expressing the target protein. The staining of the antibody is evaluated by Western blot through analyses of samples from cell lysates with and without recombinant expression of the target protein. The results show no or weak band from the unmodified cell line lysate and a strong band in the cell line with recombinant expression. Single band corresponding to the predicted size in kDa (+/-20%). Lane 1: Marker [kDa] 250, 130, 95, 72, 55, 36, 28, 17, 10 Lane 2: Negative control (vector only transfected HEK293T lysate) Lane 3: Over-expression Lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY408629) Target mass (kDa): 39.9, 37, 35.2

Antibody dilution	1:125
PROTEIN ARRAY
Validationⁱ A protein array containing 384 different antigens including the antibody target is used to analyse antibody specificity. Depending on the array interaction profile the antibody is scored as Supported, Approved, or Uncertain.	Supported Pass with single peak corresponding to interaction only with its own antigen. Antibody specificity analysis with protein arrays. Predicted and matching interactions are shown in green.

Antibody dilution	1:500
RELEVANT PUBLICATIONS
	Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter Baughman JM et al Nature 2011;476(7360):341-5
	Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals Qiu J et al Nat Commun 2013;4:2034
	The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter Pan X et al Nat Cell Biol 2013;15(12):1464-72
	Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling Logan CV et al Nat Genet 2014;46(2):188-93
	Quantitative proteomics of synaptic and nonsynaptic mitochondria: insights for synaptic mitochondrial vulnerability Stauch KL et al J Proteome Res 2014;13(5):2620-36 Application: WB
	Reconstitution of the mitochondrial calcium uniporter in yeast Kovács-Bogdán E et al Proc Natl Acad Sci U S A 2014;111(24):8985-90
	Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity Arruda AP et al Nat Med 2014;20(12):1427-35 Application: WB
	Essential role of mitochondrial Ca2+ uniporter in the generation of mitochondrial pH gradient and metabolism-secretion coupling in insulin-releasing cells Quan X et al J Biol Chem 2015;290(7):4086-96 Application: WB
	The endoplasmic reticulum mitochondrial calcium cross talk is downregulated in malignant pleural mesothelioma cells and plays a critical role in apoptosis inhibition Patergnani S et al Oncotarget 2015;6(27):23427-44 Application: IHC, WB
	Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter Lee Y et al EMBO Rep 2015;16(10):1318-33 Application: WB
	EglN2 associates with the NRF1-PGC1α complex and controls mitochondrial function in breast cancer Zhang J et al EMBO J 2015;34(23):2953-70 Application: WB
	Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals Marland JR et al J Biol Chem 2016;291(5):2080-6 Application: WB
	EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter Vais H et al Cell Rep 2016;14(3):403-410 Application: IP, WB
	Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex Tsai MF et al Elife 2016;5: Application: IP, WB
	The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1α Tosatto A et al EMBO Mol Med 2016;8(5):569-85 Application: WB
	Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle De La Fuente S et al J Biol Chem 2016;291(44):23343-23362 Application: ICC-IF
	Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether Naon D et al Proc Natl Acad Sci U S A 2016;113(40):11249-11254 Application: WB
	Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics Zampieri S et al Physiol Rep 2016;4(24): Application: WB
	Mitochondrial Ca Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity Dong Z et al Mol Cell 2017;65(6):1014-1028.e7 Application: IP, WB
	Proteolytic control of the mitochondrial calcium uniporter complex Tsai CW et al Proc Natl Acad Sci U S A 2017;114(17):4388-4393 Application: WB
	The mitochondrial Na/Ca exchanger is essential for Ca homeostasis and viability Luongo TS et al Nature 2017;545(7652):93-97 Application: WB
	High-affinity cooperative Ca binding by MICU1-MICU2 serves as an on-off switch for the uniporter Kamer KJ et al EMBO Rep 2017;18(8):1397-1411 Application: WB
	Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants Verma M et al J Neurosci 2017;37(46):11151-11165 Application: WB
	Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy Zaglia T et al Proc Natl Acad Sci U S A 2017;114(43):E9006-E9015 Application: WB
	FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation Shanmughapriya S et al Nat Commun 2018;9(1):3449 Application: WB
	The ER Stress Inducer l-Azetidine-2-Carboxylic Acid Elevates the Levels of Phospho-eIF2α and of LC3-II in a Ca-Dependent Manner Roest G et al Cells 2018;7(12): Application: WB
	Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function Sebag SC et al Exp Cell Res 2018;362(2):400-411 Application: WB
	Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses Wang X et al Front Mol Neurosci 2018;11:469 Application: ICC-IF
	MICU1 imparts the mitochondrial uniporter with the ability to discriminate between Ca and Mn Kamer KJ et al Proc Natl Acad Sci U S A 2018;115(34):E7960-E7969 Application: WB
	Restoring mitochondrial calcium uniporter expression in diabetic mouse heart improves mitochondrial calcium handling and cardiac function Suarez J et al J Biol Chem 2018;293(21):8182-8195 Application: WB
	Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice Pan M et al Am J Physiol Cell Physiol 2018;314(6):C732-C740 Application: WB
	Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria Hamilton J et al J Biol Chem 2018;293(40):15652-15663 Application: WB
	ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model Norante RP et al Int J Mol Sci 2019;20(20): Application: WB
	Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca levels and tumor growth Marchi S et al EMBO J 2019;38(2): Application: IP, WB
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment
Length (aa)	106
Antigen sequence	`HHRTVHQRIASWQNLGAVYCSTVVPSDDVTVVYQNGLPVISVRLPSRRER CQFTLKPISDSVGVFLRQLQEEDRGIDRVAIYSPDGVRVAASTGIDLLLL DDFKLV`
Matching transcripts	MCU-201 - ENSP00000349680 [100%] MCU-202 - ENSP00000362144 [100%] MCU-204 - ENSP00000440913 [96%]
Matching mouse transcripts	ENSMUSP00000020312 [94%] ENSMUSP00000029624 [43%] ENSMUSP00000122556 [23%]
ANTIGEN VIEWⁱ The Structure section provides in-house generated structures, predicted using the Alphafold source code, for the majority of the proteins and their related isoforms. Displaying protein features on the AlphaFold structures Individual splice variants can be selected in the top part of the Protein Browser (see below) and different transcript-related features such as transmembrane regions, InterPro domains and antigen sequences for antibodies can be displayed in the structure by clicking on the respective features in the Protein Browser. Clinical and population-based amino acid variants based on data from the Ensembl variation database and AlphaMissense (AM) predictions can be highlighted using the sliders to the right of the structure. These can also be used to colour the entire structure by residue index or make the structure autorotate.The structures are displayed using the NGL Viewer and can also be zoomed-in and rotated manually. The Protein Browser The ProteinBrowser displays the antigen location on the target protein(s) and the features of the target protein. Transcript names and schematic transcript structures including exons, introns and UTRs for the different isoforms are shown on top, and can be used to switch between the structures for the different splice variants. At the top of the view, the position of the antigen (identified by the corresponding HPA identifier) is shown as a green bar. A yellow triangle on the bar indicates a <100% sequence identity to the protein target. Below the antigens, the maximum percent sequence identity of the protein to all other proteins from other human genes is displayed, using a sliding window of 10 aa residues (HsID 10) or 50 aa residues (HsID 50). The region with the lowest possible identity is always selected for antigen design, with a maximum identity of 60% allowed for designing a single-target antigen (read more). The curve in blue displays the predicted antigenicity i.e. the tendency for different regions of the protein to generate an immune response, with peak regions being predicted to be more antigenic.The curve shows average values based on a sliding window approach using an in-house propensity scale. (read more). Signal peptides (turquoise) and membrane regions (orange) based on predictions using the majority decision methods MDM and MDSEC are also displayed. Low complexity regions are shown in yellow and InterPro regions in green. Common (purple) and unique (grey) regions between different splice variants of the gene are also displayed (read more), and at the bottom of the protein view is the protein scale.

MCU-201 MCU-202 MCU-204

Displaying protein features on the AlphaFold structures

The Protein Browser