Moreover, SC-101?mAb showed a higher sensitivity, thus allowing us to obtain a strong signal even at lower concentrations (Fig.?1A, panels e, f) (23.6??17.6, score 2). in basic research and as a diagnostic and prognostic tool. The specificity and biological activity of the SC-101?mAb were evaluated by Western blotting, immunofluorescence, and immunoprecipitation analyses on various cell lines. KGFR expression in breast, pancreatic, and thyroid carcinoma was assessed by immunohistochemistry (IHC) with SC-101?mAb. KGFR expression levels revealed by SC-101?mAb resulted to increase proportionally with tumor grade in breast and pancreatic cancer. In addition, SC-101?mAb was able to detect KGFR down-modulation in thyroid cancer. SC-101?mAb might represent a useful tool for basic research applications, and it AM-1638 could AM-1638 also contribute to improve the accuracy of diagnosis and prognosis of epithelial tumors. Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9773-x) contains supplementary material, which is available to authorized users. Keywords: Keratinocyte growth factor receptor, Monoclonal antibody, Breast cancer, Pancreatic adenocarcinoma, Papillary thyroid carcinoma Introduction Fibroblast growth factor receptors (FGFRs) family consists of four highly related genes, FGFR1-4, encoding proteins that are 55C72?% identical in their amino acid sequence. This family is characterized by a complexity of heterodimers and a high frequency of alternative splicing events, which justifies the signal transduction of a large number of ligands [1]. In particular, Rabbit Polyclonal to POFUT1 FGFR2 gene is subjected to an alternative splicing that generates two isoforms, the Keratinocyte growth factor (KGFR or FGFR2-IIIb) and the FGFR2-IIIc. The domains designated IIIb and IIIc, located on the third Ig loop of the FGFR2, are encoded by alternative usage of exon 8 or 9 of the FGFR2 gene. This rearrangement is cell type-dependent, since KGFR is expressed predominantly on epithelial cells, while FGFR2-IIIc is detected in cells of mesenchymal lineages. The two isoforms differ also for ligand specificity, since KGFR isoform binds with high affinity AM-1638 to FGF7/KGF, AM-1638 FGF10 and FGF22, and with low affinity to FGF1 and FGF3, while FGFR2-IIIc isoform binds to FGF1, FGF2, FGF4, FGF5, FGF6, FGF8, FGF9, FGF16, FGF17, FGF18, FGF20, FGF21, and FGF23 [2]. Given the role of both FGFR2 isoforms in inducing cell proliferation, it has been observed that their altered expression can be associated to loss of proliferation control, as documented in various types of cancer. Nevertheless, it is still controversial whether these receptors should be considered oncogenes. Indeed, they not only act as positive regulators of tumorigenesis by stimulating cell growth, but they also possess tumor suppressor properties, by enhancing cell differentiation. In fact, overexpression of FGFR2 gene has been reported in breast, lung, stomach, and pancreatic cancers [3], while its down-modulation has been demonstrated in thyroid cancer [4] and in melanoma, where FGFR2 gene can even present loss-of-function mutations [5]. Moreover, the two FGFR2 isoforms can play different roles in tumorigenesis and their effects depend upon the cell type in which they are expressed. In particular, FGFR2-IIIc expression has been correlated to epithelial to mesenchymal (EMT) transition of tumor cells in bladder cancer, which may represent a key factor in tumor progression by increasing the metastatic potential of cancer cells [6]. As for KGFR, in some cell types, its overexpression can lead to mitogenesis and tumor progression [7], but in other context it can induce cell differentiation, thus reducing the invasive potential of tumor cells [8]. The evaluation of expression and specific contribution of the two isoforms in various types of cancer is complicated by the lack of commercial antibodies that are able AM-1638 to discriminate between KGFR and FGFR2-IIIc. In fact, the only way to selectively analyze the splicing variants is to make use of specific and expensive molecular biology techniques, such as Real-Time PCR. In light of these considerations, the aim of this study was the generation and characterization.