Markham NO , Doll CA , Dohn MR , Miller RK , Yu H , Coffey RJ , McCrea PD , Gamse JT , Reynolds AB .

K01 DK092320 NIDDK NIH HHS

	FIGURE 1:. DIPA interacts specifically with p120 isoform 1. (A) Schematic of full-length DIPA, p120-1AB, and respective truncation mutants. Isoforms are named p120-1 through p120-4, depending on N-terminal start site. Designations A–D are included if the exon is present (e.g., p120-1A, p120-1BC). No letter is used if referring to multiple isoforms for which the alternatively spliced exons are either unknown or irrelevant (e.g., p120-1 isoforms). A and B, alternatively spliced exons A and B; Arm, Armadillo repeat domain; CC, p120 coiled-coil; CC1, DIPA coiled-coil 1; CC2, DIPA coiled-coil 2. (B) Growth of yeast indicates a positive interaction between human DIPA and p120. DIPA and p120 constructs were fused to the C-terminal end of the GAL4 activation domain and GAL4 DNA-binding domain, respectively. (C) Immunofluorescence of 3xFlag-DIPA (M2 anti-Flag mAb) and either endogenous p120 or exogenous human p120-1A or p120-3A (F1αSH anti-p120 pAb) in MDCK cells. wt, wild-type; p120i, p120 shRNA knockdown; p120i/p120-1A, p120 shRNA knockdown with human p120-1A rescue; p120i/p120-3A, p120 shRNA knockdown with human p120-3A rescue. Scale bars, 25 μm. (D) Western blot of immunoprecipitated lysates from p120i/p120-1A or p120i/p120-3A MDCK cells with exogenous 3xFlag-DIPA. Antibodies used for immunoprecipitation are indicated at the top of each lane. p120, pp120 mAb; α-Flag, M2 anti-Flag mAb; immunoglobulin G (IgG), KT3 isotype control mAb. Tubulin is used as a loading control for whole-cell lysate fractions (10% input). Images are representative of at least three independent experiments.
	FIGURE 2:. Endogenous DIPA colocalizes with p120, and its junctional localization requires p120 expression. (A) Immunofluorescence of endogenous DIPA (3E3 anti-DIPA mAb) and p120 (F1αSH anti-p120 pAb) in MDCK cells with either control shRNA (ctl shRNA) or p120 knockdown (p120i). (B) Immunofluorescence of endogenous DIPA (3E3 anti-DIPA mAb) and ZO-1 (anti-ZO-1 pAb) using the same cells as in A. White arrows in merged images show yellow colocalization of DIPA and p120 in A but distinctly separate localization of DIPA and ZO-1 in B. (C) The same cells shown in A viewed with confocal microscopy to show colocalization of DIPA with p120 in z-stack images (top) and a single x-y plane (bottom). White arrowheads indicate the plane in which the corresponding images were taken. (D) The same cells shown in B imaged with confocal microscopy as in C. (E) Western blot for DIPA (3E3 anti-DIPA mAb) from whole-cell lysates of MDCK with control shRNA (ctl) or p120 knockdown (p120i). Tubulin (DM1α anti–α-tubulin mAb) is shown as a loading control. (F) Immunofluorescence detection of endogenous DIPA (3E3 anti-DIPA mAb) and p120-1 (6H11 isoform-specific anti-p120 mAb) in HCA7 (top) and HEK293 (bottom) human cell lines. Scale bars, 25 μm. Images are representative of at least three independent experiments.
	FIGURE 3:. DIPA interacts with p120 family members. (A) Direct Y2H assays with full-length DIPA and Flag-ΔN-term-DIPA vs. p120-1A, p120-3A, δ-catenin, p0071, and ARVCF. Flag-ΔN-term-DIPA does not contain the first 42 amino acids of full-length DIPA. Both DIPA constructs were fused to the C-terminus of the GAL4 activation domain, and all p120 and family member constructs were fused to the C-terminus of the GAL4 DNA-binding domain. Y2H experiments using δ-catenin required the addition of 12.5 mM 3-amino-1,2,4-triazole (3-AT) to the medium to control for autoactivation. (B) Immunofluorescence detection of p120-knockdown MDCK cells with endogenous DIPA (3E3 anti-DIPA mAb) and one of five exogenous GFP fusion proteins: GFP alone, p120-3A, p120-1A, δ-catenin, or p0071. Scale bars, 25 μm. Images are representative of at least three independent experiments.
	FIGURE 4:. (A–C) DIPA family members bind to p120-1A and its family members. (A) Schematic of DIPA, Ccdc85a, and Ccdc85c proteins with structured domains and relative sizes. CC1, coiled-coil 1. CC2, coiled-coil 2; Gly-rich, glycine-rich domain. Numbers indicate amino acids. (B) ClustalW alignment of approximately the first 250 amino acids of the human DIPA family members shows at least 54% similarity and 73% identity in the coiled-coil regions. Shaded boxed areas indicate identical amino acids for at least two of the proteins. Blue and red brackets enclose the DIPA CC1 and CC2, respectively. National Center for Biotechnology Information accession numbers: Ccdc85b (DIPA), AAB05928.1; Ccdc85a, AAI31558.1; Ccdc85c, NP_001138467.1. (C) Direct yeast two-hybrid assays with full-length Ccdc85a and Ccdc85c vs. p120-1A, p120-3A, δ-catenin, p0071, and ARVCF. Both Ccdc85a and Ccdc85c were fused to the C-terminus of the GAL4 activation domain, and all p120 and family member constructs were fused to the C-terminus of the GAL4 DNA-binding domain. Y2H experiments using δ-catenin required the addition of 12.5 mM 3-AT to the medium to control for autoactivation. (D, E) Immunofluorescence detection of p120-knockdown MDCK cells with Flag-tagged Ccdc85a (D) or Flag-tagged Ccdc85c (E; M2 anti-Flag mAb) and one of five exogenous GFP fusion proteins: GFP alone, p120-3A, p120-1A, δ-catenin, or p0071. Bottom, Flag-tagged Ccdc85a (D) and Flag-tagged Ccdc85c (E; M2 anti-Flag mAb) immunofluorescence with endogenous p120 (F1αSH anti-p120 pAb) in MDCK cells. White arrows indicate junctional colocalization. Scale bars, 25 μm. Images are representative of at least three independent experiments.
	FIGURE 5:. DIPA overexpression or morpholino knockdown phenocopies an N-cadherin mutant neural tube defect in zebrafish embryos. (A) The dorsal view of AB zebrafish heads at 48 hpf shows neural tube staining with Wnt1 in situ hybridization of uninjected embryos and embryos injected with either DIPA mRNA or DIPA morpholino at the one- to two-cell stage. Embryos are oriented with anterior toward the top. Black arrowheads show closed or open neural tubes. E, eyes; L, left; R, right. Images are representative of at least three independent experiments. (B) Table showing exact numbers of embryos counted for each variable. Chi-squared tests yield p < 0.01 when comparing either mRNA or morpholino to control. (C) N-cadherin–stained cross sections of the AB zebrafish neural tube at 24–30 hpf after no injection, DIPA mRNA injection, or DIPA morpholino injection. White boxes in top row represent the borders of the zoomed images in the bottom row. E, eyes; L, left; R, right. Arrowheads, organized neuroepithelium. Arrows, disorganized cell aggregates. Scale bars, 50 μm.

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