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Chun Zeng
Chun Zeng
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Xin Yi
Xin Yi
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Danny Zipris
Danny Zipris
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Hongli Liu
Hongli Liu
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Lin Zhang
Lin Zhang
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Qiaoyun Zheng
Qiaoyun Zheng
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Krishnamurthy Malathi
Krishnamurthy Malathi
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Ge Jin
Ge Jin
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Aimin Zhou
Aimin Zhou
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
Clinical Chemistry Program, Center for Gene Regulation in Health and Diseases, Department of Cancer Biology, Barbara Davis Center of Childhood Diabetes, Central Laboratory, Department of Biological Sciences, Department of Biological Sciences, Department of Chemistry, Cleveland State University, SI 424, Cleveland, Ohio 44115, USA
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. 2008 ). RNase L is an IFN-inducible enzyme and plays an important role in the 2-5A system of IFN action against viral infection and cellular proliferation ( Silverman 1996 ). The 2-5A system consists of two enzymes: 2-5A synthetase and RNase L. IFNs
D Swolin
D Swolin
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C Brantsing
C Brantsing
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G Matejka
G Matejka
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C Ohlsson
C Ohlsson
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Abstract
Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells.
Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10−6 mol/l) decreased IGF-I mRNA levels.
In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I.
Journal of Endocrinology (1996) 149, 397–403
Trinna L Cuellar
Trinna L Cuellar
UCSF Diabetes Center, Department of Microbiology and Immunology, University of California, San Francisco, California 94122-0534, USA
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Michael T McManus
Michael T McManus
UCSF Diabetes Center, Department of Microbiology and Immunology, University of California, San Francisco, California 94122-0534, USA
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understanding of the biology of this process. Biogenesis of miRNAs Mature miRNAs are derived from two major processing events, driven by sequential cleavages by the RNAse-III enzymes Drosha and Dicer (Fig. 1 ) (for comprehensive reviews on
T M Lovell
T M Lovell
School of Animal and Microbial Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK
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P G Knight
P G Knight
School of Animal and Microbial Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK
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R T Gladwell
R T Gladwell
School of Animal and Microbial Sciences, The University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK
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was subsequently purified as described in the standard Tri-reagent protocol. The final RNA pellet was resuspended in 100 μl nuclease-free water (containing RNA Secure; Ambion), and then treated with RNase-free DNase (15 min at 37 °C; RQ1; Promega). The
K Alexander H Iwen
K Alexander H Iwen
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Oezge Senyaman
Oezge Senyaman
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Arne Schwartz
Arne Schwartz
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Maren Drenckhan
Maren Drenckhan
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Britta Meier
Britta Meier
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Dirk Hadaschik
Dirk Hadaschik
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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Johannes Klein
Johannes Klein
Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
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have been published. Stimulation of 3T3-L1 cells with MCs has been reported to cause an increase in cAMP concentrations ( Boston & Cone 1996 , Norman et al . 2003 ). ACTH and αMSH inhibit leptin expression and secretion in 3T3-L1 cells and primary rat
C Doyon
C Doyon
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada
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V L Trudeau
V L Trudeau
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada
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T W Moon
T W Moon
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada
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(Campellcroft, ON, Canada). They were acclimated to the laboratory for at least 6 weeks in 1275 l fiberglass tanks of well-aerated dechloraminated City of Ottawa tap water at 13 ± 1 °C with a constant 12-h light/12-h dark photoperiod. Holding tanks initially
T M Lovell
T M Lovell
School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK
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P G Knight
P G Knight
School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK
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R T Gladwell
R T Gladwell
School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK
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Tri-reagent protocol. The final RNA pellet was resuspended in 100 μl nuclease-free water (containing RNA Secure (Ambion, Huntington, Cambridgeshire, UK)), then treated with RNase-free DNase (15 min at 37 °C; RQ1; Promega). The purified RNA was re
G Tachas
G Tachas
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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S Lofthouse
S Lofthouse
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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C J Wraight
C J Wraight
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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B F Baker
B F Baker
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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N B Sioufi
N B Sioufi
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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R A Jarres
R A Jarres
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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A Berdeja
A Berdeja
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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A M Rao
A M Rao
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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L M Kerr
L M Kerr
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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E M d’Aniello
E M d’Aniello
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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M J Waters
M J Waters
Antisense Therapeutics Ltd, Level 1, 10 Wallace Ave, Toorak, Victoria 3142, Australia
Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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Isis Pharmaceuticals Inc., 1986 Rutherford Ave, Carlsbad, California 92008, USA
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
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maintained to allow RNAse H activity. Controls CoA and CoB are standard control oligonucleotides used in in vitro oligonucleotide selection, while controls CoC and CoD were developed following selection of the optimised sequence. They represent a scrambled
Laura Fozzatti
Laura Fozzatti
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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María L Vélez
María L Vélez
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Ariel M Lucero
Ariel M Lucero
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Juan P Nicola
Juan P Nicola
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Iván D Mascanfroni
Iván D Mascanfroni
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Daniela R Macció
Daniela R Macció
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Claudia G Pellizas
Claudia G Pellizas
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Germán A Roth
Germán A Roth
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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Ana M Masini-Repiso
Ana M Masini-Repiso
Centro de Investigaciones en Bioquímica Clínica e Inmunología-(CIBICI) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, 5000 Córdoba, Argentina
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–III) have been well characterized, which are widely expressed in most cell types. The NOS-mediated reaction involves l -arginine oxidation with generation of NO and l -citrulline ( Nathan & Xie 1994 , Guix et al. 2005 ). One of the physiologically more
Carla Brancia
Carla Brancia
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Paola Nicolussi
Paola Nicolussi
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Pietro Cappai
Pietro Cappai
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Giorgio La Corte
Giorgio La Corte
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Roberta Possenti
Roberta Possenti
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Gian-Luca Ferri
Gian-Luca Ferri
NEF-Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Cagliari, Italy
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Sassari, Italy
Department of Neuroscience, ‘Tor Vergata’ University, Rome, Italy
Istituto Zootecnico Caseario della Sardegna, Bonassai, Sassari, Italy
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PubMed
antero-posterior, vertical slices (2–3 mm thick), immersion fixed in paraformaldehyde (40 g/l in 0.1 mol/l PO 4 buffer, pH 7.2, 0–4 °C for 3 h), then washed in phosphate buffered saline (PBS: 10 mol/l PO 4 , pH 7.2–7.4, 150 mmol/l NaCl) containing
