Localization of a human fibroblast extracellular protein in cells and tissues by monoclonal antibody

McLean, W. H. Irwin; Orchin, J C; Foster, Heather; Fogarty, Barbra; Nevin, N. C.

doi:10.1042/bst0180279

Biochemical Society Transactions

1990

DOI: 10.1042/bst0180279

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Localization of a human fibroblast extracellular protein in cells and tissues by monoclonal antibody

W. H. Irwin McLean

J C Orchin

Heather Foster

et al.

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1991

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“…&is antibody recognises a 140kDa extracellular lycoprotein secreted by human fibroblasts. The exact knction of this protein is unknown, but tissue distribution suggests a structural role in connective tissues [6,7]. Synthesis and secretion of this protein appears as normal in fibroblasts from DEB individuals.…”

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Protein analysis in Epidermolysis Bullosa

McLean

McKenna

Anderson

et al. 1991

Biochemical Society Transactions

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Epidermol sis Bullosa (EB) is an inherited disorder where a enetic dezct leads to abnormal blistering of the skin. d e r e are a number of forms of the disease which vary both in pattern of inheritance and in pheno e [1,2]. The precise enetic defect is unknown in all cases, P owever this is ve kikely to lie in an extracellular matrix component or a cetr adhesion molecule of skin. Here, we present early results of protein analysis of cultured skin fibroblasts from patients with the more severe dystrophic form of the disease (DEB).Fibroblasts were cultured from skin biopsies of individuals with DEB, and unaffected individuals of similar age by the explant method. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal calf serum (FCS) and antibiotics (Gibco). For immunofluorescence staining, cells were grown ernight on 10-well Multiwell slides (Flow) at a densi of l~cells/well in DMEM su plemented with 20% FCS. 8 ells were washed in phosphate {uffered saline and fiied in acetone for 10 min prior to staining with neat supernatant monoclonal antibodies and a 1:40 dilution of rabbit-anti-mouse IgG TRITC conjugate (Sigma). All monoclonal antibodies were made in house &3] . Collagen secretion was assayed from cultures of 6x10 cells over 72 hour y a colourimetric extracellular proteins were analysed by 2D-PAGE as described elsewhere [5]. Cells of similar passage number were used for all comparisons.The results of immunofluorescence staining with antibodies against selected candidate roteins are shown in Fig.1. Staining with a monoclonal antigody against fibronectin, the major cell adhesion molecule of fibroblasts is shown in Fig.lA. Internal staining of secretory vesicles is visible and the accumulation of fibronectin extracellularly ap ears as normal (stainin of normal controls not shown).gainin with monoclonaf antibody 1.4D1 is shown in Fig.lB. &is antibody recognises a 140kDa extracellular lycoprotein secreted by human fibroblasts. The exact knction of this protein is unknown, but tissue distribution suggests a structural role in connective tissues [6,7]. Synthesis and secretion of this protein appears as normal in fibroblasts from DEB individuals. In addition to those shown here, a further six monoclonal antibodies produced against uncharacterised fibroblast cell surface and extracellular antigens were used to stain normal and DEB fibroblasts. In every case, a normal pattern of staining was observed.performed using 2D-PAGE. This system allows simultaneous detection and quantitation of about 50 groups of extracellular proteins, of which onl fibronectin, metalloproteinases I, I1 and I11 and d M P are known with certainty [8]. In early comparisons of normal and DEB fibroblast cell lines, no ma'or change in the synthesis or electrophoretic mobility o! these extracellular proteins has been found (results not shown). The secretion of collagen by normal and DEB fibroblast cell lines was also assayed and no significant differences have been found. The results reported here show that t...

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confidence: 97%

Protein analysis in Epidermolysis Bullosa

McLean

McKenna

Anderson

et al. 1991

Biochemical Society Transactions

Self Cite

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Localization of a human fibroblast extracellular protein in cells and tissues by monoclonal antibody

Cited by 1 publication

References 0 publications

Protein analysis in Epidermolysis Bullosa

Protein analysis in Epidermolysis Bullosa

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