SIX1 - Antibodies - The Human Protein Atlas


	Antibody HPA001893	Antibody CAB058690

ANTIBODY INFORMATION
Provider	Atlas Antibodies Sigma-Aldrich	Atlas Antibodies
Product name	HPA001893	AMAb90544
Host species	Rabbit	Mouse
Clonalityⁱ The antibodies are designated mAB for monoclonal and pAb for polyclonal.	pAb	mAb
Concentration	0.07 mg/ml	Not known
Purity	Affinity purified using the PrEST-antigen as affinity ligand	Protein A/G
Released in versionⁱ The release of the Human Protein Atlas in which the antibody was first published.	1.2	11.0
Referencesⁱ References to publications in which the antibody has been used.	26	1
Proper citation	Atlas Antibodies Cat#HPA001893, RRID:AB_1079991	Atlas Antibodies Cat#AMAb90544, RRID:AB_2665581
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	N/A ICC IHC WB N/A 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 and nucleoli.	N/A

Antibody dilution	Human assay: A-431 fixed with PFA, dilution: 1:35 Human assay: U-251MG fixed with PFA, dilution: 1:35 Human assay: U2OS fixed with PFA, dilution: 1:35
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.	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Parathyroid gland RNA expression: 46.2 nTPM LOW EXPRESSION Liver RNA expression: 0.0 nTPM	Enhanced - Orthogonal Antibody staining mainly consistent with RNA expression data across 44 tissues. HIGH EXPRESSION Parathyroid gland RNA expression: 46.2 nTPM LOW EXPRESSION Liver RNA expression: 0.0 nTPM

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	HIER pH6
Antibody dilution	1:50	1:250
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 extensive gene/protein characterization data.	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.	High consistency between antibody staining and RNA expression data.	High 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. Band of predicted size in kDa (+/-20%) with additional bands present. 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, LY401814) Target mass (kDa): 32.2	Uncertainⁱ 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. No bands detected. Analysis performed using a standard panel of samples. 250 130 100 70 55 35 25 15 10

Antibody dilution	1:250	1:500
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.	N/A

Antibody dilution	1:3000
RELEVANT PUBLICATIONS
	Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer Wan F et al Int J Cancer 2008;123(1):32-40	Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals Washausen S et al Biol Open 2018;7(6): Application: IHC
	Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition McCoy EL et al J Clin Invest 2009;119(9):2663-77
	The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-beta signaling Micalizzi DS et al J Clin Invest 2009;119(9):2678-90
	Eya2 is required to mediate the pro-metastatic functions of Six1 via the induction of TGF-β signaling, epithelial-mesenchymal transition, and cancer stem cell properties Farabaugh SM et al Oncogene 2012;31(5):552-62
	SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation Ono H et al Oncogene 2012;31(47):4923-34
	The miR-106b-25 cluster targets Smad7, activates TGF-β signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer Smith AL et al Oncogene 2012;31(50):5162-71
	Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways Iwanaga R et al Breast Cancer Res 2012;14(4):R100
	Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration Le Grand F et al J Cell Biol 2012;198(5):815-32
	Persistently altered epigenetic marks in the mouse uterus after neonatal estrogen exposure Jefferson WN et al Mol Endocrinol 2013;27(10):1666-77 Application: IHC
	3D mouse embryonic stem cell culture for generating inner ear organoids Koehler KR et al Nat Protoc 2014;9(6):1229-44 Application: IHC
	Six homeoproteins and a Iinc-RNA at the fast MYH locus lock fast myofiber terminal phenotype Sakakibara I et al PLoS Genet 2014;10(5):e1004386 Application: ChIP, WB
	Overexpression of sineoculis homeobox homolog 1 predicts poor prognosis of hepatocellular carcinoma Kong J et al Int J Clin Exp Pathol 2014;7(6):3018-27 Application: ICC-IF, WB
	Axud1 Integrates Wnt Signaling and Transcriptional Inputs to Drive Neural Crest Formation Simões-Costa M et al Dev Cell 2015;34(5):544-54 Application: IHC
	The homeoprotein SIX1 controls cellular senescence through the regulation of p16INK4A and differentiation-related genes Adrados I et al Oncogene 2016;35(27):3485-94 Application: ChIP, ICC-IF, IHC, WB
	The Six1 oncoprotein downregulates p53 via concomitant regulation of RPL26 and microRNA-27a-3p Towers CG et al Nat Commun 2015;6:10077 Application: WB
	SIX1 Oncoprotein as a Biomarker in a Model of Hormonal Carcinogenesis and in Human Endometrial Cancer Suen AA et al Mol Cancer Res 2016;14(9):849-58 Application: IHC, WB
	SIX1 coordinates with TGFβ signals to induce epithelial-mesenchymal transition in cervical cancer Sun SH et al Oncol Lett 2016;12(2):1271-1278 Application: IHC, WB
	Six1 homeoprotein drives myofiber type IIA specialization in soleus muscle Sakakibara I et al Skelet Muscle 2016;6(1):30 Application: IHC
	Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells Roellig D et al Elife 2017;6: Application: IHC
	Six1 expression is associated with a poor prognosis in patients with glioma Zhang X et al Oncol Lett 2017;13(3):1293-1298 Application: IHC
	Increased Six1 expression is associated with poor prognosis in patients with osteosarcoma Chao L et al Oncol Lett 2017;13(5):2891-2896 Application: IHC
	A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages Tchieu J et al Cell Stem Cell 2017;21(3):399-410.e7 Application: ICC-IF
	Six1 is essential for differentiation and patterning of the mammalian auditory sensory epithelium Zhang T et al PLoS Genet 2017;13(9):e1006967 Application: IHC
	Respective contribution of the cephalic neural crest and mesoderm to SIX1-expressing head territories in the avian embryo Fonseca BF et al BMC Dev Biol 2017;17(1):13 Application: IHC
	Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression Zhang L et al Nat Commun 2018;9(1):1047 Application: WB
	SIX1 represses senescence and promotes SOX2-mediated cellular plasticity during tumorigenesis De Lope C et al Sci Rep 2019;9(1):1412 Application: ChIP
ANTIGEN INFORMATION
Antigen	Recombinant protein fragment	Recombinant protein
Length (aa)	141
Antigen sequence	`CFKEKSRGVLREWYAHNPYPSPREKRELAEATGLTTTQVSNWFKNRRQRD RAAEAKERENTENNNSSSNKQNQLSPLEGGKPLMSSSEEEFSPPQSPDQN SVLLLQGNMGHARSSNYSLPGLTASQPSHGLQTHQHQLQDS`
Matching transcripts	SIX1-205 - ENSP00000494686 [100%]
Matching mouse transcripts	ENSMUSP00000059026 [99%] ENSMUSP00000024947 [62%] ENSMUSP00000125169 [43%]
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.

SIX1-205

Displaying protein features on the AlphaFold structures

The Protein Browser