PMC:546208 / 18911-21897 JSONTXT

{"project":"2_test","denotations":[{"id":"15649318-14681682-9187503","span":{"begin":1763,"end":1765},"obj":"14681682"}],"text":"Development and characterization of Scrib antibodies\nTo analyze expression and intracellular distribution of Scrib in cultured cells, we prepared a Scrib-specific antibody (Scrib-472), as described in the Methods section. The Scrib-472 antibody recognized a protein of an apparent molecular mass of more than 200 kDa in a number of different cell extracts (Fig. 3A). Scrib was easily detected in the epithelial cell lines 293 and MDCKII, in the fibroblast cell line CV-1, and also in the T lymphocyte cell line Jurkat (Fig. 3A). These results indicate that our antibody recognizes Scrib-proteins of different species, being human (Jurkat and 293), monkey (CV-1) and dog (MDCKII). The Scrib-472 antibody also reacted with an Xpress-hScrib fusion protein produced in 293T cells transfected with the corresponding DNA (Fig. 3B). In Fig. 3B, lane 2, which depicts a Western analysis of untransfected 293T cell lysate with Scrib-antibodies, no band of endogenous Scrib is seen. Longer exposure, however, did show a band indicating that Scrib is expressed in these cells, however, 293T cells express much lower levels of endogenous Scrib as compared to 293 cells (our unpublished observations). The Scrib protein was migrating slower in SDS gels than would be expected from its theoretically calculated molecular mass (175 kDa). Possible explanations include anomalous migration per se, and posttranslational modifications. To investigate whether the Scrib-472 antibody not only recognizes denatured Scrib protein on Western blots but also is capable of detecting Scrib in fixed cells, MDCKII cells were grown to confluency on glass coverslips and stained with the Scrib-472 antibodies. From previous studies, it is known that Scrib is localized in cell-cell contacts [17]. As shown in Fig. 3C, the Scrib-472 antibody indeed is capable to detect native Scrib in cell-cell contacts in fixed cells.\nFigure 3 Characterization of anti-Scrib antibodies. (A) Total cell extracts were prepared from the following cell lines: human embryonic kidney epithelial cells (293) (lane 1), dog normal kidney epithelial cells (MDCK) (lane 2), human T lymphocytes (Jurkat) (lane 3), and African green monkey kidney fibroblast cells (CV-1) (lane 4). Approximately 30 μg of protein from each extract was analysed by SDS-PAGE and Western blotting with the Scrib-472 antibodies. The position of molecular markers are as shown. (B) Total cell extracts of 293T cells, either not transfected (lane 2), or transiently transfected with Xpress-hScrib that is composed of the full length human Scrib protein fused to an Xpress-epitope-tag at its amino-terminus (lanes 1 and 3) were analyzed by SDS-PAGE and Western blotting with an anti-Xpress antibody (lane 1) or with the Scrib-472 antibody (lanes 2 and 3). The position of molecular markers are as shown. (C) MDCKII cells, grown on glass coverslips, were fixed and stained with Scrib-472-antibodies. Immunofluorescence was visualized by epifluorescence microscopy.\n\nS"}