Asterisks in cCf (corresponding asterisks in cCf) indicate nuclear regions. Other antibodies used for immunofluorescence were as follows: undiluted culture supernatant from 9E10 hybridomas; and FITC- or TRITC-labeled secondary antibodies (Jackson Laboratories; 1:100). cDNA Library Screening and DNA Sequencing An expression library of oocyte in gt11 (cat. no. ZL5000b; CLONTECH Laboratories) was screened with antiserum C532. 106 plaques were screened, and three positive phages giving strong immunoreactivity contained the same insert after digestion of DNA with EcoRI. The cDNA insert contained an internal EcoRI site, giving one fragment of 1 1.2 kb and one fragment of 4.0 kb. These two fragments were subcloned into pSK+ and pKS+, respectively, and the resulting plasmids (called D902 and D898) were used to prepare constructs either by direct subcloning or PCR amplification. The nucleotide sequence of the cDNA inserts was decided on both strands by cycle sequencing using FS polymerase (Perkin-Elmer). The 1,248-bp fragment (D902) contained a 114-bp noncoding region followed by a 1,134-bp open reading frame. The 3,948-bp fragment (D898) contained a 2,976-bp open reading frame followed by a 972-bp noncoding region. It was concluded that in the original cDNA insert, the 1.2-kb fragment must be located 5 with respect to the 4.0-kb fragment. The two fragments joined by the EcoRI site formed a 4,104-bp-long open reading frame. Protein Sequence Analysis Cingulin sequence was analyzed with Kyte-Doolitle (DNAStrider1.3) and MacStripe programs (Knight 1994) (http://www.york.ac.uk/depts/biol/web/homes.htm) to generate plots of predicted hydrophobicity and coiled-coil structure, respectively. PSORT and ScanProsite programs (http:// expasy.hcuge.ch) were used to identify sorting Rabbit Polyclonal to IKK-alpha/beta (phospho-Ser176/177) signals and other sequence features, and BLAST version Monomethyl auristatin E 2.0 was used to identify homologies with other proteins. The heptad-containing region in the amino acid sequence of cingulin was delineated by hand rather than by computer since the former technique is more sensitive to locating discontinuities that may exist in the heptad phasing. Secondary structure analysis was carried out using the Robson (Garnier et al. 1978) and Chou-Fasman techniques (Chou and Fasman 1978). Potential interchain ionic interactions between charged residue pairs in positions 2e-1g, 1g-2e, Monomethyl auristatin E 2a-1g, 1g-2a, 1e-1d, and 1d-1e were calculated as a function of relative chain stagger and chain polarity (McLachlan and Stewart 1975; Parry et al. 1977). Cell Culture and Preparation of Cell Lysates Mammalian cells were cultured in a humidified incubator at 37C and 6% CO2. MDCK II (a gift of Dr. K. Simons, European Molecular Biology Laboratory, Heidelberg, Germany) and 9E10 hybridoma cells (a gift of Dr. G. Evan, Imperial Cancer Research Fund, London) were grown in DME supplemented with 2 mM glutamine and 10% FBS (Hyclone), CaCo2 cells (a gift Monomethyl auristatin E of Dr. A. LeBivic, University of Marseille, Marseille, France) in DME supplemented with 2 mM glutamine, 20% FBS, 1% nonessential amino acids, 5 U/ml penicillin/streptomycin; insect cells (Sf21) at 30C in liquid culture in TC100 supplemented with 10% FBS, and 5 U/ml penicillin/streptomycin. To prepare lysates for glutathione-S-transferase (GST) pull-down assays, confluent epithelial monolayers or Sf21 cells (30 h after infection at an initial density 0.5 106 cells/ml with mouse Monomethyl auristatin E ZO-1 virus stock, virus provided by Dr. M. Itoh, Kyoto University, Kyoto, Japan) were rinsed twice with cold PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and were lysed in lysis-buffer-triton (LBT: 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, 5 g/ml leupeptin, 5 g/ml antipain, 5.