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	Figure 1. The nucleotide and derived amino acid sequence of Xenopus laevis cingulin. Single underline indicates nuclear localization sequence. Dashed underline indicates predicted coiled-coil sequence. The sequences are available from GenBank/EMBL/DDBJ under accession number AF207901.
	Figure 2. Northern blot analysis of Xenopus tissues total RNA, using cingulin and actin probes. An ∼5.4-kb cingulin message is detected in intestine (epithelial tissue) but not in fat bodies (connective tissue). Numbers on the left indicate the migration of RNA size markers.
	Figure 4. Electron microscopic analysis of purified chicken cingulin rod. (A) Rotary-shadowed molecules of cingulin rod. The arrow indicates one example of a 130-nm-long and 2-nm-wide molecule. The arrowheads indicate one example of an apparent cingulin oligomer. (B) Negatively stained twisted tangles of cingulin rod. The average length of the twisted tangles was 432 ± 51 nm (n = 36). Only isolated aggregates were scored. Bars, 150 nm.
	Figure 3. Structure predictions and domain organization of Xenopus laevis cingulin. (A) Kyte-Doolittle hydrophobicity plot of cingulin. Amino acid positions are indicated on the x-axis. (B) Coiled-coil prediction analysis of cingulin using the MacStripe program. A value of 1 indicates highest probability of coiled-coil. Horizontal lines above the plot indicate the seven frames of the heptad repeats. (C) A schematic domain organization of Xenopus laevis cingulin (see also Fig. 10). (D) Diagram of GST fusion protein constructs used in pull-down assays. (E) Diagram of Myc-tagged cingulin constructs used in transfection experiments.
	Figure 6. Interaction of cingulin with ZO-1, ZO-2, ZO-3, myosin, AF-6, and symplekin. (A) GST pull-downs from epithelial cell lysates using bacterially expressed cingulin. Western blots of MDCK cell lysates after affinity purification on glutathione-Sepharose beads attached to GST, GST-XC(1-378), or GST-XC(377-1,368), or before affinity purification (lysate). ZO-1, ZO-2, and AF-6 appear to associate only with cingulin NH2-terminal fragment, myosin associates with both NH2-terminal and COOH-terminal fragments, and symplekin does not associate with any fragment. (B) GST pull-downs from reticulocyte lysates containing radioactively labeled in vitro translated proteins. Autoradiography of 35S-labeled ZO-1, ZO-2, ZO-3, and myosin, after affinity purification on glutathione-Sepharose beads attached to GST (GST), GST fusions of Xenopus cingulin NH2-terminal fragment (GST-XC(1-378)), or COOH-terminal fragment (GST-XC(377-1,368)). ZO-1 and ZO-2 associate only with the NH2-terminal fragment, ZO-3 and myosin associate with both NH2-terminal and COOH-terminal fragments. (C) Coimmunoprecipitation of cingulin with ZO-1 and ZO-2. Western blot analysis, using anticingulin antiserum C532, of cingulin in Triton-soluble (CaCo-T) and SDS-soluble (CaCo-S) fractions of human CaCo2 cultured epithelial cells. (top left) Cell extract; (top right) extract after immunoprecipitation with anticingulin antiserum (IP α-cing); (bottom left) extract after immunoprecipitation with anti–ZO-1 antibodies (IP αZO-1); and (bottom right) extract after immunoprecipitation with anti–ZO-2 antibodies (IP αZO-2). Cingulin (Mr 140 kD) is detected in ZO-1 and ZO-2 immunoprecipitates from the SDS-soluble fraction. Polypeptides of Mr 100–135 kD detected in the Triton-soluble fraction of CaCo2 lysates probably represent proteolytic degradation products.
	Figure 5. Self-interaction of Xenopus laevis cingulin. Radioactively labeled full-length cingulin, generated by in vitro transcription/translation, was detected by autoradiography after affinity purification on glutathione-Sepharose beads attached to GST, or GST-XC(377-1,368), GST-XC(1-378), or GST fused to a human ZO-1 fragment (amino acids 849–1,060) (GST-HuZO-1(849-1,060)). Strong association was detected with the COOH-terminal fragment of cingulin, no association with ZO-1 fragment or GST. Numbers on the left indicate approximate molecular sizes in kilodaltons, based on the migration of prestained markers.
	Figure 8. Localization of cingulin in transiently transfected MDCK cells. Double immunofluorescent staining of mock-transfected MDCK cells (a and a′), or MDCK cells transfected with XC(1-1,368)-myc (b and b′), or with XC(1-378)–myc (c, c′, d, and d′), or with XC(377-1,368)-myc (e, e′, f, and f′), using anticingulin antiserum C532 followed by TRITC anti-rabbit (a–f) and anti–myc 9E10 antibody followed by FITC anti-mouse (a′–f′). C532 labels endogenous cingulin and transfected cingulin when the COOH-terminal fragment is present in the transfection construct. 9E10 labels expressed cingulin. Mock-transfected cells were transfected with solvent or with vector alone. Single arrow in b (corresponding arrow in b′) indicates colocalization of endogenous and transfected cingulin in the junctional region between two cells. Double arrows in b and b′ indicate a junction containing endogenous cingulin but not transfected cingulin. Asterisks in c–f (corresponding asterisks in c′–f′) indicate nuclear regions. Asterisks in d′ and in e and e′ indicate cytoplasmic labeling of transfected protein. Thin arrows in f and f′ indicate brightly stained cytoplasmic aggregates. Bar, 10 μm.
	Figure 9. Localization of cingulin in Xenopus laevis embryos (gastrula stage) after microinjection of early embryos with cingulin constructs in pCS2. Double immunofluorescent staining of mock-microinjected embryos (a and a′), or embryos microinjected with XC(377-1,368)-myc in pCS2 (b and b′), using anticingulin antiserum R902 followed by FITC anti-rabbit (a and b) and 9E10 antibody followed by TRITC anti-mouse (a′ and b′). Arrows in a and b (corresponding arrow in b′) indicate junctional cingulin immunolabeling. Star in b′ (corresponding star in b) indicates cytoplasmic labeling of transfected protein. Asterisk in b′ indicates nuclear region. Note that XC(377-1,368)–myc is distributed in the cytoplasm and absent from junctions and nuclear regions. c and d show two different focal planes (c, apical; d, subapical) of an embryo that was microinjected with residues 1–378 of cingulin without Myc tag (XC(1-378)) and stained with anticingulin antiserum R902 followed by TRITC-anti-rabbit. R902 antiserum was raised against the NH2-terminal region of cingulin (Cordenonsi et al. 1997), and recognizes both endogenous and transfected cingulin. White arrows in c indicate apical junctional cingulin staining in two adjacent blastomeres. Black arrows in d indicate nuclear regions in a lower plane of focus in the same blastomeres shown in c. Black star in d indicates cytoplasm. Black dots visible in b′ and c against the light background represent pigment granules. Bar, 20 μm.
	Figure 10. (A) A schematic cartoon showing the putative organization of cingulin as a parallel dimer with globular and coiled-coil domains. (B) Proposed names, sizes, and amino acid boundaries of different Xenopus cingulin regions. (C) Summary of in vitro interactions of GST fusion proteins of NH2-terminal (1–378) and COOH-terminal (377–1,368) fragments of Xenopus cingulin. Positive score indicates interaction detected (with number of + proportional to relative intensities of signal using different approaches), a negative score indicates interaction not detected.

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