Home » AP-1 » Lloyd Klickstein (Brigham and Women’s Hospital, Boston, MA)

Lloyd Klickstein (Brigham and Women’s Hospital, Boston, MA)

Lloyd Klickstein (Brigham and Women’s Hospital, Boston, MA). avidity of E7 for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Therefore, despite its dissimilarity to known integrin ligands, the specific molecular interaction shown here shows that E-cadherin is definitely a direct counter receptor for the E7 integrin. The cadherins constitute a family of cell surface adhesion molecules that are involved in calcium- dependent homophilic cell to cell adhesion (Takeichi, 1990). The best studied human being cadherins, E-, P-, N-, and VE-cadherin, have a restricted cells distribution: E- and P-cadherin are indicated in epithelial cells (Nose and Takeichi, 1986; Shimoyama et al., 1989(Beverly, MA). Oligonucleotides were from Oligotech (Boston, MA). Additional chemicals were purchased from (St. Louis, MO). ICAM-1CFc (the entire extracellular region of human being ICAM-1 10-Oxo Docetaxel fused to the hinge and Fc portion of human being IgG1) was a nice gift of Dr. Lloyd Klickstein (Brigham and Women’s Hospital, Boston, MA). Purified human being IgG1 was from (La Jolla, CA). mAbs The mAb used (all mouse IgG against human being antigens) were as follows: E4.6 (antiCE-cadherin, IgG1; Cepek et al., 1994), HECD-1 (antiCE-cadherin, IgG1; Shimoyama et al., 1989Sverige, Uppsala, Sweden). The columns were washed with TBS and 1 mM CaCl2, pH 7.4, and then eluted with 0.2 M glycine and 1 mM CaCl2, pH 2.3. Fractions comprising purified fusion proteins were dialyzed into TBS and 1 mM CaCl2, pH 7.4 and then stored at ?20C. The purity of fusion protein was assessed by SDS-PAGE and Coomassie blue staining, and the concentration was determined by Bradford assay using BSA as a standard (Bio-Rad Labs., Hercules, CA). Production of Soluble 35S-labeled Recombinant E7 Integrin Soluble recombinant E7 was produced by COS-7 cells after transient transfection using DEAE-dextran (Coligan et al., 1994, Unit 10-14) with the plasmids pAPRM8/tEs and pAPRM8/t7s. Control transfections were carried out with the antisense constructs pAPRM8/tEas and pAPRM8/t7as. After incubation for 48 h in total medium, the cells were washed once with PBS and then 1 mCi 35S-Express (San Francisco, CA) in 100 l TBS, pH 7.4, and blocked while described above before addition of Fc fusion proteins. IEL or transfected JY cells were labeled with BCECF-AM (Molecular Probes, Eugene, OR) as previously explained (Cepek et al., 1993). During labeling of JY cells, 10% (vol/vol) heat-inactivated normal human being serum was included to block Fc receptors. Adhesion assays were carried out in 0.1% BSA and HBS with mixtures of MnCl2, MgCl2, CaCl2, or 1 mM EGTA, as indicated (see text). In antibody obstructing experiments, cells or wells were preincubated with mAbs for 10 min at PCDH8 4C as explained 10-Oxo Docetaxel in the text. For cell activation experiments, cells were preincubated with antibodies or 50 ng/ml PMA at 4C for 15 min. Adhesion assays were carried out 10-Oxo Docetaxel as explained previously (Cepek et al., 1993) with the following modifications. Labeled cells were brought into contact with the microtiter plate wells by centrifugation at 60 for 2 min (IEL) or 1 min (JY). After incubation at 37C for 10 min, nonadherent cells were removed by washing with 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, and HBS at 37C unless the effect of divalent cations was being assessed, in which case HBS only was used. Since in these assays the fluorescence of input cells was quenched to some degree by the presence of adhesion buffer, but the percent bound was identified after eliminating the buffer, some apparent readings of 100% are acquired. Homophilic adhesion assays were carried out as explained above with the following modifications. 16E6.A5 cells were released from culture dishes using 0.02% (wt/vol) trypsin, 2 mM CaCl2, and HBS to minimize proteolysis of cadherins. After adding 2 vol of 0.04% (wt/vol) soy bean trypsin inhibitor, HBS, and washing twice with HBS, the cells 10-Oxo Docetaxel were resuspended in 0.1% BSA, HBS, and 1 mM CaCl2 and allowed to settle.