title: Distinct expression of C4.4A in colorectal cancer detected by different             antibodies.
abstract: The metastasis-associated gene C4.4A encodes a glycolipid-anchored membrane             protein expressed in several human malignancies. The present study aimed to perform             a detailed assessment of C4.4A expression in colorectal cancer tissues, in terms             of intra-cellular localization, intra-tumoral location and difference in molecular             weight. To advance this goal, we developed three new antibodies against the C4.4A             protein (two polyclonal Abs: C4.4A-119 and C4.4A-277 and one monoclonal Ab: C4.4A             GPI-M) to use in addition to the two previously produced polyclonal Abs (C4.4A-81,             C4.4A GPI-P). Antibody specificities were confirmed by absorption tests. Western             blot analysis and immunohistochemistry showed that the C4.4A-119 and C4.4-277             Abs detected 70-kDa C4.4A, mainly in the cytoplasm, irrespective of intra-tumoral             location. The C4.4A GPI-P and C4.4A GPI-M Abs reacted with the membranous ~40-kDa             C4.4A, exclusively at the tumor invasive front, and each detected an identical             tumor cell population. The tested antibodies showed varied C4.4A detection rates             in 33 CRC tissues. The C4.4A-277 Ab yielded the highest positive rate in 29 of             33 CRC tissues (87.9%), while the C4.4A GPI-P and C4.4A GPI-M Abs each only showed             33.3% positivity. The present findings suggest that the GPI anchor signaling sequence             may be essential for detecting membranous C4.4A at the invasive front of CRC tissues.
authors.affiliation: Department of Surgery, Gastroenterological Surgery, Osaka University, Suita, Osaka, Japan. hyamamoto@gesurg.med.osaka-u.ac.jp
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Ryota
authors.initials: R
authors.lastname: Oshiro
authors.forename: Masahisa
authors.initials: M
authors.lastname: Ohtsuka
authors.forename: Mamoru
authors.initials: M
authors.lastname: Uemura
authors.forename: Naotsugu
authors.initials: N
authors.lastname: Haraguchi
authors.forename: Junichi
authors.initials: J
authors.lastname: Nishimura
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takemasa
authors.forename: Tsunekazu
authors.initials: T
authors.lastname: Mizushima
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Doki
authors.forename: Masaki
authors.initials: M
authors.lastname: Mori
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002462:Cell Membrane / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; C115145:C4.4 monoclonal antibody; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; C472589:LYPD3 protein, human; D010446:Peptide Fragments