METTL14 - Antibodies - The Human Protein Atlas


	Antibody HPA038002

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich
Product name	HPA038002
Host species	Rabbit
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb
Concentration	0.2612 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.	7.0
Referencesⁱ References to publications in which the antibody has been used.	21
Proper citation	Atlas Antibodies Cat#HPA038002, RRID:AB_10672401
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 A-431 shows localization to nucleoplasm.

Antibody dilution	Human assay: A-431 fixed with PFA, dilution: 1:130 Human assay: U-251MG fixed with PFA, dilution: 1:130 Human assay: U2OS fixed with PFA, dilution: 1:130
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.	Supportedⁱ 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 testis shows strong cytoplasmic and nuclear positivity in cells in seminiferus ducts. Testis

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	1:800
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.	Consistent with 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.	Medium 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.	Supportedⁱ The staining of an antibody is evaluated by Western Blot through analysis of samples from different cell lysates. A supportive score is given if band(s) of predicted size in kDa (+/-20%) is detected. Band of predicted size in kDa (+/-20%) with additional bands present. Analysis performed using a standard panel of samples. 250 130 95 72 55 36 28 17 10

Antibody dilution	1:250
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.	Approved Pass with quality comment low specificity (binding to 1-2 antigens >15% and <40%). Antibody specificity analysis with protein arrays. Predicted and matching interactions are shown in green.

Antibody dilution	1:3000
RELEVANT PUBLICATIONS
	A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation Liu J et al Nat Chem Biol 2014;10(2):93-5
	N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells Wang Y et al Nat Cell Biol 2014;16(2):191-8
	N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions Liu N et al Nature 2015;518(7540):560-4 Application: WB
	Dynamic m(6)A mRNA methylation directs translational control of heat shock response Zhou J et al Nature 2015;526(7574):591-4 Application: ICC-IF, WB
	The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells Lin S et al Mol Cell 2016;62(3):335-345 Application: WB
	N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression Tirumuru N et al Elife 2016;5: Application: WB
	N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein Liu N et al Nucleic Acids Res 2017;45(10):6051-6063 Application: WB
	Region-specific RNA mA methylation represents a new layer of control in the gene regulatory network in the mouse brain Chang M et al Open Biol 2017;7(9): Application: IHC, WB
	Thiol-linked alkylation of RNA to assess expression dynamics Herzog VA et al Nat Methods 2017;14(12):1198-1204 Application: WB
	Dynamic m6A modification regulates local translation of mRNA in axons Yu J et al Nucleic Acids Res 2018;46(3):1412-1423 Application: ICC-IF
	N-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications Wang Y et al Nat Neurosci 2018;21(2):195-206 Application: IHC
	METTL3-mediated m6A modification is required for cerebellar development Wang CX et al PLoS Biol 2018;16(6):e2004880 Application: WB
	METTL3 regulates WTAP protein homeostasis Sorci M et al Cell Death Dis 2018;9(8):796 Application: WB
	The mA-methylase complex recruits TREX and regulates mRNA export Lesbirel S et al Sci Rep 2018;8(1):13827 Application: IP
	Cross-talk among writers, readers, and erasers of mA regulates cancer growth and progression Panneerdoss S et al Sci Adv 2018;4(10):eaar8263 Application: WB
	The N-methyladenosine (mA)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells Vu LP et al Nat Med 2017;23(11):1369-1376 Application: IP, WB
	METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA mA Modification Weng H et al Cell Stem Cell 2018;22(2):191-205.e9 Application: IP, WB
	N-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response Zhou J et al Mol Cell 2018;69(4):636-647.e7 Application: WB
	Mettl3-mediated mRNA mA methylation promotes dendritic cell activation Wang H et al Nat Commun 2019;10(1):1898 Application: WB
	Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets Zhang F et al Hum Mol Genet 2018;27(22):3936-3950 Application: WB
	mA mRNA Methylation Regulates Human β-Cell Biology in Physiological States and in Type 2 Diabetes De Jesus DF et al Nat Metab 2019;1(8):765-774 Application: IHC, WB
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment
Length (aa)	95
Antigen sequence	`RSWNMDSRLQEIRERQKLRRQLLAQQLGAESADSIGAVLNSKDEQREIAE TRETCRASYDTSAPNAKRKYLDEGETDEDKMEEYKDELEMQQDEE`
Matching transcripts	METTL14-201 - ENSP00000373474 [96%]
Matching mouse transcripts	ENSMUSP00000029759 [92%] ENSMUSP00000087848 [92%] ENSMUSP00000134198 [92%] ENSMUSP00000141542 [28%] ENSMUSP00000119664 [26%]
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.

METTL14-201

Displaying protein features on the AlphaFold structures

The Protein Browser