Tang T K, Mazzucco C E, Benz E J, Jr, Marchesi V T

Tang T K, Mazzucco C E, Benz E J, Jr, Marchesi V T. domain (GAL4-DB) in vector pAS2-1 (Clontech). This construct was used as bait to screen a human lymphocyte cDNA library fused to a GAL4 activation domain (GAL4-AD) in the pACT2 vector (Clontech). The two types of plasmids were then cotransformed into Y190, and the transformants were selected on SD minimal medium as previously described (40). Positive colonies were further tested for -galactosidase activity using a colony-lift assay and liquid assay as described by the manufacturer (Clontech). Mouse monoclonal to EphA4 To narrow down the head domain region of 4.1R (4.1R-HD) that binds to CPAP, constructs containing various portions of 4.1R-HD were fused to GAL4-DB of the pAS2-1 vector (Fig. ?(Fig.1A).1A). The C terminus of CPAP (residues 897 to 1338) was subcloned into the pACT2 vector. Yeast cells (Y187) were simultaneously transformed with the above two constructs and assayed for -galactosidase activity using a colony-lift assay or liquid assay as described above. Open in a separate window FIG. 1 4.1R interacts with CPAP in a yeast two-hybrid system. The clone was first isolated by a yeast two-hybrid screen from a human lymphocyte cDNA library using the head domain (residues 1 to 209) of 4.1R-135 as bait (4.1R-HD). (A) Mapping the region of 4.1R-135 that interacts with CPAP. The constructs containing various portions of fused in-frame to the DNA-binding domain were cotransformed with a clone that expressed CPAP (residues 897 to 1338) fused to the activation domain of reporter gene using a colony-lift assay is shown. The column on the right represents the liquid assay for -galactosidase (-gal) activity using ONPG as a substrate. (B) Schematic drawing of the overlapping cDNA clones that span the entire coding region of CPAP. Isolation of CPAP cDNA clones and Northern blot analysis. The initial cDNA clone (Q1) isolated by yeast two-hybrid screen was used as a probe to screen a human testis cDNA library (Clontech). Several overlapping cDNA clones that cover the entire coding region of were obtained (Fig. ?(Fig.1B).1B). The conditions for screening and DNA sequencing were described previously (46). All DNA sequencing data were compiled and analyzed using the GCG software programs of the Wisconsin Sequence Analysis Package. For RNA analysis, a blot filter (Clontech) with 2 g of polyadenylated RNA from multiple human tissues was hybridized with a 32P-labeled cDNA probe (nucleotides [nt] 2899 to 3423) as previously described (46). The same probe was stripped and reprobed with -actin cDNA to quantify RNA loading. Antibody production. Polyclonal antibodies against CPAP and the head domain of 4.1R-135 (anti-HD-4.1R) were raised in rabbits. The cDNAs encoding the C-terminal region of CPAP (cCPAP; residues 1070 to 1338) and the head domain (HD; residues 55 to 198) of 4.1R-135 were fused in frame to glutathione-was subcloned in-frame into a cytomegalovirus promoter-driven FLAG epitope-tagged expression vector. SiHa cells (5 106) were transiently transfected with 10 g of FLAG-tagged plasmid as previously described (40). For Western blot Cefotiam hydrochloride analysis, the cell extracts prepared from the indicated cells or tissues were separated by sodium dodecyl sulfateC7.5% Cefotiam hydrochloride polyacrylamide gel electrophoresis (SDS-PAGE), blotted Cefotiam hydrochloride onto a PVDF membrane, and probed with the antibodies indicated in Fig. ?Fig.55 as previously described (40). The immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Pierce, Rockford, Ill.). Open in a separate window FIG. 5 Direct association of 4.1R-135 with CPAP in vivo and in vitro. (A) Characterization of anti-CPAP and anti-N-4.1R antibodies. The production of antibodies against the C terminus of CPAP (anti-CPAP, a polyclonal antibody) and the N-terminal head domain of 4.1R-135 (anti-N-4.1R MAb) is described in the text. SiHa cells were transiently transfected with a FLAG-tagged CPAP plasmid. The cell lysates (50 g) prepared from mouse testis, untransfected cells (SiHa and Molt4), and transfected SiHa cells, as indicated, were separated by SDS-PAGE and immunoblotted with anti-CPAP (lanes 1 to 4), anti-FLAG (lane 5), or anti-N-4.1R (lanes 6 and 7) antibodies. (B) Direct association of 4.1R-135 with CPAP in vivo. The cell lysates prepared from SiHa cells were immunoprecipitated (IP) with anti-N-4.1R MAb (lane 1) or a nonrelevant MAb (H25B10, lane 2). The immunoprecipitated protein complexes were then analyzed by immunoblotting (IB) with anti-CPAP antibody. Furthermore, the cell lysates prepared from.