HE4 (whey acidic protein (WAP)-type four-disulfide core-2, WFDC2) is a small secretory protein that may influence sperm maturation. HE4 gene expression is high in pulmonary epithelial cells and in some ovarian cancers. HE4 protein has a WAP motif that contains eight cysteines forming four disulfide bonds at the core of the protein. The WAP motif functions as a protease inhibitor in many of the family members that contain them.
Background References
1. Markovic-Lipkovski J et al. Variable Expression of Neural Cell Adhesion Molecule Isoforms in Renal Tissue: Possible Role in Incipient Renal Fibrosis. PLoS One 10:e0137028 (2015).
2. Zhuang H et al. Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells. Mol Cancer 13:243 (2014).
Tissue Specificity
Expressed in a number of normal tissues, including male reproductive system, regions of the respiratory tract and nasopharynx. Highly expressed in a number of tumors cells lines, such ovarian, colon, breast, lung and renal cells lines. Initially described as being exclusively transcribed in the epididymis.
Western blot analysis of HE4 on different lysates with Rabbit anti-HE4 antibody (ET1702-61) at 1/2,000 dilution.
Lane 1: SW480 cell lysate (15 µg/Lane) Lane 2: HepG2 cell lysate (15 µg/Lane) Lane 3: OVCAR-3 cell lysate (15 µg/Lane) Lane 4: Mouse lung tissue lysate (20 µg/Lane) Lane 5: Mouse kidney tissue lysate (20 µg/Lane)
Predicted band size: 13 kDa Observed band size: 20 kDa
Exposure time: 1 minute;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1702-61) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of HepG2 cells labeling HE4 with Rabbit anti-HE4 antibody (ET1702-61) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HE4 antibody (ET1702-61) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of HepG2 cells labeling HE4.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1702-61, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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