Naked2 (NKD2) is a person in the Naked family and negatively

Naked2 (NKD2) is a person in the Naked family and negatively regulates canonical Wnt signaling. member of the Naked family and negatively regulates canonical Wnt signaling.(1,2) NKD2, with a molecular mass of 50?kDa, is predominantly localized around the vesicles or plasma Thiazovivin membrane.(3) Full-length NKD2 and its 1C331 fragment are poorly soluble. NKD2 contains an N-terminal catalytic domain name (1C300 residues), a proline-rich domain name, and a C-terminal ER targeting domain. The C-terminus of NKD2 is usually highly disordered. The N-terminal region of NKD2 behaves as an intrinsically unstructured protein but contains most of the NKD2 functional domains, including myristoylation, EF-hand motif, Dishevelled binding region, vesicle recognition, and membrane targeting motif.(3) NKD2 binds to multiple proteins and may function as a switch protein through its several functional motifs.(3) NKD2 is an inducible antagonist of canonical Wnt signaling and has been showed to act by binding and inactivating Dishevelled, a positive regulator of Wnt signaling.(4,5) Wnt signaling is usually involved in virtually every aspect of embryonic development. It controls homeostatic self-renewal in a number of adult tissues,(6) and its dysregulation contributes to oncogenesis.(7) This allows the prediction that NKD2 may play a role in embryo development and tumor formation by affecting Wnt signaling. It is also reported that myristoylated NKD2 interacts with the cytoplasmic C-terminal fragment of a Golgi processed form of TGF, coats TGF-containing exocytic vesicles, and escorts these vesicles to the basolateral membrane of polarized epithelial cells.(8,9) FEN-1 The information above implies that NKD2 may have some potential and important functions, such Thiazovivin as affecting cell signaling pathway and malignancy development. In order to conduct further studies around Thiazovivin the structure activity relationship, protein detection, and cell-signaling pathway of NKD2, our laboratory established a monoclonal antibody against NKD2 (anti-NKD2 MAb). Materials and Methods Reagents RPMI 1640 and fetal bovine serum were purchased from GibcoBRL (Grand Island, NY). Freund’s adjuvant (total and incomplete), HAT medium, goat anti-mouse IgG-labeled peroxidase, and polyethylene glycol 1450 (PEG) were from Sigma (St. Louis, MO). O-phenylenediamine (OPD) and enhanced chemiluminescence (ECL) were purchased from Sangon (Shanghai, China). rProtein A Sepharose column was purchased from GE Healthcare (Stockholm, Sweden). Materials The recombinant NKD2 protein (NKD21-217) was expressed and purified as explained previously.(3) SP2/0 cell collection, human colorectal carcinoma SW480 cell collection and LOVO cell collection, and BALB/c mice (8 weeks aged, female) were obtained from the Cancer Research Center of Xiamen University (Xiamen, China). Mice immunization NKD21-217 was used to raise antibodies in four BALB/c mice. Each mouse was first immunized with 40?g NKD21-217 (in 0.2?mL phosphate buffer saline [PBS]) fully emulsified with 0.2?mL Freund’s complete adjuvant by subcutaneous injection. After 2 weeks, each mouse was given a booster dose of 20?g NKD21-217 in incomplete Freund’s adjuvant. The booster injection was repeated two times at 2-week intervals and blood was collected for serum after the last immunization. The serum was monitored for antibody titers against NKD21-217 by indirect ELISA. Mice with sustained antibody titers above 1104 were selected and intravenously injected with 20?g NKD21-217 without Freund’s adjuvant 3 days before cell fusion. The indirect ELISA was performed as follows(10): 10?g/mL NKD21-217 in covering buffer (0.05?M bicarbonate, pH 9.6) was coated in the plates overnight at 4C. The Thiazovivin plates were blocked with 5% skim milk at 37C for 2?h and washed with PBST (PBS containing 0.05% Tween-20) three times. The supernatants of serum or hybridoma cell culture were incubated in the plates for 1?h at 37C. After washing, goat anti-mouse IgG-labeled peroxidase was incubated and added for 1?h in 37C. OPD with 0.03% hydrogen peroxide was put into develop color as well as the optical density (OD) was measured at 490?nm with a microplate audience (model 680, Bio-Rad, Tokyo, Japan). Establishment of hybridomas We followed a cell.