mAb B4/7 was derived from a mouse immunized with a protein fraction isolated from detergent extracts of MDCK cells using fusion proteins containing the cytoplasmic domain of low density lipoprotein receptor. Binding of GEF-H1 to this fusion protein was not specific. Hybridoma production and subcloning were as described (Hauri et al., 1985 (link)). Five subclones of the originally isolated hybridoma line were isolated and found to recognize all the same protein in immunoblots and to produce the same pattern in immunofluorescence experiments.
All peptides and antipeptide antibodies were produced by Gramsch Laboratories. The following peptides were used to generate rabbit polyclonal antibodies: anti–cGEF-H1 NH2 terminus, MSRIESLTRARTERC; anti–cGEF-H1 COOH terminus, CDFTRMQDIPEETES; anti–cGEF-H1 alternative domain, CRGHDRLDLSVTIRSVH; anti–ZO-1, YTDQELDETLNDEVC; and anti–claudin-4, PRTDKPYSAKYSAAC. The peptides were conjugated to epoxy-activated Sepharose (Amersham Biosciences), and the antibodies were affinity purified as described (Balda et al., 1996 (link)). For α-tubulin, mAb 1A2 was used (Kreis, 1987 (link)), and in some immunofluorescence experiments ZO-1 was detected with rat monoclonal R40.76 (Anderson et al., 1988 (link)) or with a rabbit polyclonal antibody (Sheth et al., 1997 (link)). α-Catenin was detected with the M12K rabbit polyclonal antibody (Herrenknecht et al., 1991 (link)). GTPases were detected by immunoblotting using the following anti-GTPase antibodies: Rho, rabbit polyclonal antibody sc-179 anti-RhoA (Santa Cruz Biotechnology, Inc.) and Rac1, mouse mAb 102 (BD Transduction Laboratories).
All peptides and antipeptide antibodies were produced by Gramsch Laboratories. The following peptides were used to generate rabbit polyclonal antibodies: anti–cGEF-H1 NH2 terminus, MSRIESLTRARTERC; anti–cGEF-H1 COOH terminus, CDFTRMQDIPEETES; anti–cGEF-H1 alternative domain, CRGHDRLDLSVTIRSVH; anti–ZO-1, YTDQELDETLNDEVC; and anti–claudin-4, PRTDKPYSAKYSAAC. The peptides were conjugated to epoxy-activated Sepharose (Amersham Biosciences), and the antibodies were affinity purified as described (Balda et al., 1996 (link)). For α-tubulin, mAb 1A2 was used (Kreis, 1987 (link)), and in some immunofluorescence experiments ZO-1 was detected with rat monoclonal R40.76 (Anderson et al., 1988 (link)) or with a rabbit polyclonal antibody (Sheth et al., 1997 (link)). α-Catenin was detected with the M12K rabbit polyclonal antibody (Herrenknecht et al., 1991 (link)). GTPases were detected by immunoblotting using the following anti-GTPase antibodies: Rho, rabbit polyclonal antibody sc-179 anti-RhoA (Santa Cruz Biotechnology, Inc.) and Rac1, mouse mAb 102 (BD Transduction Laboratories).
