caa a22 4500 397557787 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199605 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021339 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021339 Expression of the Cholera Toxin B Subunit in the Golgi Apparatus of Swiss 3T3 Cells Inhibits DNA Synthesis Induced by Basic Fibroblast Growth Factor [Elektronische Daten] [Yasuhiro Hashimoto, Akihiro Oshima, Hisashi Narimatsu, Akemi Suzuki] We attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection. Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human β1,4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector. The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression. The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluo-resence microscopy; and third, detection of GM1 binding activity in cell lysates. Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis. These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and platelet-derived growth factor. Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites. These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding. © by the Journal of Biochemistry Regular Papers nationallicence basic fibroblast growth factor nationallicence cDNA transfection nationallicence cholera toxin B subunit nationallicence DNA synthesis nationallicence ganglioside nationallicence Hashimoto Yasuhiro Departments of Membrane Biochemistry The Tokyo Metropolitan Institute of Medical Science 3-18 -22 Honkomagome, Bunkyo-ku, Tokyo 113 aut Oshima Akihiro Departments of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113 aut Narimatsu Hisashi The Institute of Life Science, Soka University 1-236 Tangi-cho Hachioji, Tokyo 192 aut Suzuki Akemi Departments of Membrane Biochemistry The Tokyo Metropolitan Institute of Medical Science 3-18 -22 Honkomagome, Bunkyo-ku, Tokyo 113 aut The Journal of Biochemistry Oxford University Press 119/5(1996-05), 985-990 0021-924X 119:5<985 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021339 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021339 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hashimoto Yasuhiro Departments of Membrane Biochemistry The Tokyo Metropolitan Institute of Medical Science 3-18 -22 Honkomagome, Bunkyo-ku, Tokyo 113 aut NATIONALLICENCE 700 1- Oshima Akihiro Departments of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113 aut NATIONALLICENCE 700 1- Narimatsu Hisashi The Institute of Life Science, Soka University 1-236 Tangi-cho Hachioji, Tokyo 192 aut NATIONALLICENCE 700 1- Suzuki Akemi Departments of Membrane Biochemistry The Tokyo Metropolitan Institute of Medical Science 3-18 -22 Honkomagome, Bunkyo-ku, Tokyo 113 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/5(1996-05), 985-990 0021-924X 119:5<985 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford