(2001) Nature 409, 211C215 [PubMed] [Google Scholar] 38

(2001) Nature 409, 211C215 [PubMed] [Google Scholar] 38. from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively. to remove insoluble material, prior to pulldown assays. At least two biological repeats were performed for each experimental set. Yeast Two-hybrid Screen The globular head domain of human paracingulin (residues 1C603) was fused C-terminally to LexA in the pB27 vector. This construct was used to screen a human placenta library in the presence of 0.5 mm 3-amino-1,2,4-triazole (Hybrigenics, Paris, France). A high confidence conversation (based on the PIM biological score) (37) was detected with four unique clones of human ZO-1, made up of sequences between nucleotides 5468 and 7049, corresponding to residues 1455C1736 of ZO-1. A very high confidence conversation was detected with five unique clones of human PLEKHA7, made up of sequences between nucleotides 1868 and 2321, corresponding to residues 602C892 of human PLEKHA7. Cell Culture and Transfection MDCK cells, Caco-2 cells, and Rat1 fibroblasts were cultured in DMEM (made up of pyruvate for Caco-2 cells) supplemented with FBS, 100 models/ml penicillin, 100 g/ml streptomycin, and 1 minimal essential medium (MEM), nonessential amino acids. MDCK and Rat1 cell lines expressing either full-length or N-terminal deletions (1C110, 1C209) of YFP-tagged canine CGNL1 (generated in the vector pTRE2Hyg) were obtained by transfection with Lipofectamine 2000 and selection in hygromicin. mpkCCDc14 cells were cultured as explained (35). MDCK cell clones depleted of PLEKHA7 were obtained by transfection of wild-type MDCK cells with the pTER vector, made up of an insert designed to target the sequence(s) AACCTGCCAAGTGACTACAAGT and ATCGCAGTCACGAGGATTCCTT. Stable transfectants were generated by selection in zeocin (29), and individual clones were isolated by cloning rings. Stable MDCK cell lines depleted of ZO-1, ZO-2, or p120ctn were gifts of Dr. A. Fanning (University or college of North Carolina) (13) and Dr. A. Reynolds (Vanderbilt University or college) (38), respectively. Immunoprecipitation and Immunoblotting For immunoprecipitations, cells Foxo1 were washed twice in ice-cold PBS and lysed in coimmunoprecipitation buffer (150 mm NaCl, 20 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mm EDTA, and total protease inhibitor) for 15 min at 4 C. Lysates were clarified by centrifugation for 15 min at 13,000 rpm. Antibodies (5 l of rabbit serum anti-PLEKHA7, anti-CGN, anti-CGNL1, and preimmune sera, 2 g of anti-p120ctn 15D2 and 8D11) were coupled with 20 l of pre-washed G-protein Dynabeads (Invitrogen) (1 h at 4 C) and then incubated with either mpkCCDc14 or MDCK lysates (16 h at 4 C). Beads were washed in coimmunoprecipitation, and proteins were eluted by boiling in JNJ-26481585 (Quisinostat) SDS sample buffer and analyzed by SDS-PAGE and immunoblotting. At least three biological repeats were performed for each experimental set. Immunofluorescence Microscopy Cells on coverslips were fixed with JNJ-26481585 (Quisinostat) chilly methanol for 10 min at ?20 C, washed with PBS, incubated with main antibody (1 h at 30 C), washed, incubated with secondary antibody (30 min at 37 C), and mounted with Vectashield medium (Reactolab). Specimens were analyzed with Axiovert S100 or Zeiss 510 META microscopes. For semiquantitative analysis of junctional labeling, we compared the junctional labeling of CGN with afadin, and CGNL1 with E-cadherin, reference proteins whose junctional localization was not affected by either ZO-1 or ZO-2 depletion. Five confocal images for each double-immunolabeled sample were analyzed with ImageJ software. Pixel intensity for each channel was JNJ-26481585 (Quisinostat) measured in the selected junctional area, and the averaged background signal was subtracted. Relative transmission was expressed as a ratio between CGN/afadin and CGNL1/E-cadherin, calculated over the five different images for each sample. At least three biological repeats were performed for each experiment. Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR) was used to assess mRNA expression in MDCK cell clones. Total RNA was prepared using the RNeasy mini kit (Qiagen), retrotranscribed using iScript cDNA synthesis kit (Bio-Rad), and analyzed by SYBR Green-based PCR as explained (39). The following primers were used: PLEKHA7, forward 5-ATT GCT CAC CAG CAG AGG TT-3 and reverse 5-TTC CAG GCA TTT TCC ATC TC-3; CGNL1 forward 5-CTC AAG GAC CTG GAA TAC GAG C-3 and reverse 5-TCC GAG AGC AAA TCC GAG TT-3; Hypoxantine-guanine phosphoribosyltransferase forward 5-TGG ACA GGA CTG AGC GGC-3 and reverse 5-TGA GCA CAC AGA GGG CTA CG-3. (conversation: very strong,.