A Monoclonal Antibody Produced Against a Rat Esophageal Carcinoma Cell Line Reacts with an Integrin-Like Molecule Expressed by Rat Epithelial Cells

Abstract

A monoclonal antibody, designated MAb-5A IIgG1), was generated against a tumorigenic rat esophageal epithelial cell line, B-2T. MAb-5A reacted with a series of non-tumorigenic and tumorigenic epithelial cell lines derived from F-344 rat esophagi or tracheas. However, the highest level of antigen expression was detected on tumorigenic rat epithelial cell lines. A trace amount of antigen was detected in primary cultures of normal rat epithelial cells derived from either esophagi or tracheas. MAb-5A did not react with rat fibroblasts. MAb-5A reacted with B-2T derived tumor tissues propagated in vivo but showed only slight reactivity with normal rat esophageal epithelial tissues. Cell surface radioiodination, extraction and immunoprecipitation experiments were conducted to characterize the antigen molecule. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed two protein bands with mobilities corresponding to approximately 140 and 120 kDa, respectively. Under reducing conditions the 140 kDa band shifted to approximately 120 kDa whereas the 120 kDa band shifted upward to approximately 130 kDa. The non-reduced/reduced mobilities of these bands suggest that they may be members of the integrin family of matrix receptors. A polyclonal antibody to the beta 1 subunit immunoprecipitated two similar bands detected by MAb-5A, further suggesting that the antigen complex may be related to members of the integrin superfamily.

The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans

“…Cells were incubated in mouse anti-rat α1β1 integrin receptor IgG2a (10 μg/ml, Millipore) at room temperature for 1 hour, rinsed 3× with PBS, rinsed once with blocking solution, incubated for 1 hour in goat anti-mouse IgG (H&L) AF488-antibody (1:500 v/v Invitrogen). For α6β1 labeling, cells were incubated in mouse anti-rat α6β1 integrin receptor IgG1 (1:200 v/v, Millipore) [28] at room temperature for 1 hour, rinsed 3× with PBS, rinsed once with blocking solution, then incubated for 1 hour in goat anti-mouse IgG1 AF488-antibody (1:500 v/v, Invitrogen).…”

A disintegrin and metalloprotease 21 (ADAM21) is associated with neurogenesis and axonal growth in developing and adult rodent CNS

“…Other primary antibodies were against ␣6␤1 integrin (immunogen: rat esophageal carcinoma cell line B2T, mouse ascites fluid, IgG1, 1:3,000, MAB1410, Chemicon [Jamasbi et al, 1992]), vimentin (immunogen: vimentin purified from bovine lens, protein-A-purified mouse IgG2a, clone 3B4, 1:1,000, CBL202, Chemicon [Bohn et al, 1992]), glial fibrillary acidic protein (GFAP; immunogen: purified glial filament, chromatographic columnpurified mouse IgG1, clone G-A-5, 1:1,000, MAB3402, Chemicon [Debus et al, 1983]), NeuN (immunogen; purified cell nuclei from mouse brain, purified mouse IgG1, 1:1,000, MAB377, Chemicon [Mullen et al, 1992]), neuronal class III ␤tubulin (protein-A-purified mouse IgG2a, clone Tuj1, 1:3,000, MMS-435P, Babco, Richmond, CA [Lee et al, 1990]), olfactory marker protein (OMP; goat IgG, 1:500, a gift from Dr. F. Margolis, University of Maryland, Baltimore, MD [Keller and Margolis, 1975;Baker et al, 1989]), p75 neurotrophin receptor (192-IgG, mouse IgG1, 1:100, a gift from Dr. Eugene Johnson, Jr., Washington University, St. Louis, MO [Chandler et al, 1984]), rat endothelial cell antigen-1 (RECA-1, immunogen: stromal cells from rat lymph node, tissue culture supernatant, mouse IgG1, HIS52, 1:100, MCA970, Serotec, Raleigh, NC [Duijvestijn et al, 1992]), TIMP1 (ascites fluid purified by protein-G chromatography, mouse IgG1, 102D1, 1:100, MAB13429, Chemicon [Hurskainen et al, 1996]), TIMP2 (purified mouse IgG1/k, 67-4H11, 1:500, MAB3310, Chemicon [Fujimoto et al, 1993[Fujimoto et al, , 1995), TIMP3 (purified mouse IgG1/k, 136-13H4, 1:500, MAB3318, Chemicon [Fariss et al, 1997]), and TIMP4 (peptide affinity-purified rabbit IgG, 1:300, AB816, Chemicon). As a positive control for ADAM21, sections through the testis were also processed for immunostaining.…”