Journal:

Article Title: Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

doi: 10.1073/pnas.0407974101

Figure Lengend Snippet: Generation of EBP50-deficient mice. (A) Targeted disruption of the EBP50 locus. A fragment of the wild-type locus (Top) containing the first and last two exons (solid boxes) and the polyadenylation signal (pA) of EBP50 is shown. The targeting construct (Middle) consisting of a 4.0-kb 5′ genomic fragment, a 1.8-kb 3′ genomic fragment, and the neoR gene without the polyadenylation signal (NeoΔpA) replaces a 16-kb genomic fragment containing exons 1–4 of the EBP50 gene in the targeted mutated locus (Bottom). The solid arrowheads flanking the neoR gene represent LoxP sites. The positions of the Southern blot probe (hatched boxes), EcoRI sites (E), and the EcoRI wild-type and mutated genomic fragments (double-pointed arrows) are shown. The Southern analysis is shown beneath. (B) Schematic representation of the EBP50 domain structure. The two PDZ domains (1 and 2) and the C-terminal ERM-binding (EB) region are shown with the respective boundaries in amino acids. Solid arrowheads indicate EBP50 mRNA splice sites that delimit EBP50's six exons. Some known interacting proteins for the various domains are listed in italics above each domain. The dimerization with itself or NHERF2 and the interaction with NHE3 most likely extend through both PDZ domains. (C) Western blot analysis of EBP50 from whole organs homogenized in TNN buffer. The organs were collected from two mice per each genotype.

Article Snippet: The genomic DNA of the NHERF1/EBP50 gene was obtained by screening a 129/SvJ mouse genomic DNA bacterial artificial chromosome library (Research Genetics) with a probe derived by PCR from exon 1 of mouse full-length EBP50 cDNA (IMAGE EST clone 2192738 [American Type Culture Collection (ATCC)].

Techniques: Construct, Southern Blot, Binding Assay, Western Blot

Journal:

Article Title: Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

doi: 10.1073/pnas.0407974101

Figure Lengend Snippet: Decreased expression levels of ERM proteins in EBP50(–/–) BBM. (A) Schematic protocol of BBM preparation. The protein concentration was measured in each boxed fraction, and samples containing 30 μg of proteins [except from S4 (supernatant 4), which contained only 1.5 μg of proteins] were loaded on polyacrylamide gels. TL, total tissue lysates. P4 (pellet 4) corresponds to the BBM fraction. (B) Western blot analysis of kidney and small intestine BBM fractionation samples from EBP50 (+/+) and (–/–) mice was performed with the indicated antibodies. The quantification analysis (Right) was performed with the imagej program (National Institutes of Health) and shows the expression levels of ezrin and NHE3 normalized to vinculin in kidney fractions. For ezrin, only the upper bands, which represent ezrin, were quantified. Similar results were obtained at least seven times for BBM prepared from either male or female mice. (C–F) The expression of phosphorylated ezrin was analyzed with phospho-ERM antibody by immunofluorescence of kidney (C and D) and jejunum (E and F) sections from EBP50(+/+) (C and E) and (–/–) (D and F) 5-week-old littermates. The samples were imaged with a Zeiss LSM 510 confocal microscope by using the HeNe 543-nm laser and identical imaging parameters. Note the higher apical expression of phosphorylated ezrin (arrows) in wild-type kidney tubules and intestinal villi.

Article Snippet: The genomic DNA of the NHERF1/EBP50 gene was obtained by screening a 129/SvJ mouse genomic DNA bacterial artificial chromosome library (Research Genetics) with a probe derived by PCR from exon 1 of mouse full-length EBP50 cDNA (IMAGE EST clone 2192738 [American Type Culture Collection (ATCC)].

Techniques: Expressing, Protein Concentration, Western Blot, Fractionation, Immunofluorescence, Microscopy, Imaging

Journal:

Article Title: Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

doi: 10.1073/pnas.0407974101

Figure Lengend Snippet: The EB region of EBP50 is cleaved in BBM. (A) The Western blot analysis of EBP50 in BBM fractions from EBP50(+/+) and (–/–) mice was carried out on the same samples as in Fig. 2B. Note the presence of EBP50 full-length and low-MW products in membrane fractions from EBP50(+/+) mice. (B)(Upper) EBP50 splice isoforms (I1 to I4) are schematically drawn, and the corresponding size in amino acids is indicated on the left. The black bar indicates the PCR used to identify the isoforms, and the arrowhead marks the position of the EcoRI site used for restriction-fragment-length polymorphism. The PCR on reverse transcribed mRNA extracted from various human tissues and cell lines is shown (Lower Left). The PCR fragments corresponding to the isoforms indicated on the left were subcloned from the U937 PCR product (Middle Right) and analyzed for RFLP with EcoRI (Bottom Right). FL, control full-length EBP50. (C) Pull-down assay with GST ezrin-NT and Npt2-CT fusion proteins (5 μg) of proteins (300 μg) from pooled P3 and P4 EBP50(+/+) or (–/–) kidney BBM fractions. Thirty micrograms of proteins from P3 and P4 fractions are included (TL). Note the specific precipitation of EBP50 FL (arrowhead) by ezrin-NT and of low-MW products (bracket) by Npt2-CT. (D) Western analysis showing EBP50 FL (arrow) and low-MW products (bracket) in human tissue lysates (huTL) in comparison with mouse P4 kidney fraction.

Article Snippet: The genomic DNA of the NHERF1/EBP50 gene was obtained by screening a 129/SvJ mouse genomic DNA bacterial artificial chromosome library (Research Genetics) with a probe derived by PCR from exon 1 of mouse full-length EBP50 cDNA (IMAGE EST clone 2192738 [American Type Culture Collection (ATCC)].

Techniques: Western Blot, Pull Down Assay

Journal:

Article Title: Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

doi: 10.1073/pnas.0407974101

Figure Lengend Snippet: EBP50 interacts with phosphorylated ERM proteins. (A) Coimmunoprecipitation of phosphorylated ERM proteins (P-ERM) with EBP50 from EBP50(+/+) and (–/–) kidney BBM P4 fractions. Full-length EBP50, FL and arrowhead; cleavage products, bracket. (B) Gel-filtration analysis of protein complexes from EBP50(+/+) kidney BBM P4 fractions with antibodies indicated on the left. Arrowhead indicates FL EBP50 and TL, total lysate. Note coelution of phosphorylated ERM proteins with FL EBP50 in a low-MW peak and with actin in a high-MW peak.

Article Snippet: The genomic DNA of the NHERF1/EBP50 gene was obtained by screening a 129/SvJ mouse genomic DNA bacterial artificial chromosome library (Research Genetics) with a probe derived by PCR from exon 1 of mouse full-length EBP50 cDNA (IMAGE EST clone 2192738 [American Type Culture Collection (ATCC)].

Techniques: Filtration

Journal:

Article Title: Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia

doi: 10.1073/pnas.0407974101

Figure Lengend Snippet: Morphology of intestinal villi in EBP(–/–) mice. (A and B) periodic acid/Schiff reagent (PAS) staining of intestinal villi from EBP50(+/+)(A) and (–/–)(B) littermates showing a higher number of PAS+ goblet cells in (–/–) villi. (×100.) (C) Transmission electron microscopy of ileum epithelial cells from 5-week-old littermates shows EBP50(+/+) ordered, rod-like microvilli emerging from a structured terminal web region (bracket) contrasting with the EBP50(–/–) disorganized microvilli and thick terminal web. (×12,000.)

Article Snippet: The genomic DNA of the NHERF1/EBP50 gene was obtained by screening a 129/SvJ mouse genomic DNA bacterial artificial chromosome library (Research Genetics) with a probe derived by PCR from exon 1 of mouse full-length EBP50 cDNA (IMAGE EST clone 2192738 [American Type Culture Collection (ATCC)].

Techniques: Staining, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

doi: 10.7554/eLife.58464

Figure Lengend Snippet: ( a ) Cryo-EM data processing scheme. The resulting maps from the final heterogeneous refinement are superimposed for comparison (cyan and brick). ( b ) Orientation distribution of particles for the final 3D reconstruction. ( c ) The gold standard FSC curve for the final map. ( d ) Local resolution estimation of the final map colored in rainbow.

Article Snippet: The following datasets were generated: Tsutsumi N Jude KM Gati C Garcia KC 2020 Structure of human Frizzled5 by fiducial-assisted cryo-EM Electron Microscopy Data Bank EMD-21927 Tsutsumi N Jude KM Gati C Garcia KC 2020 Structure of human Frizzled5 by fiducial-assisted cryo-EM RCSB Protein Data Bank 6WW2

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

doi: 10.7554/eLife.58464

Figure Lengend Snippet: ( a ) Cryo-EM map (gray) and structural model of hFzd5 ICL3 BRIL in detergent complexed with anti-BRIL Fab and anti-Fab Nb. The model is colored as follow: the sequences from Fzd5 (cyan), BRIL and the linker between BRIL and Fzd5 (light blue), Fab heavy chain (pink), Fab light chain (light pink) and Nb (brown). ( b ) The map-model FCS curve generated by Phenix refinement (masked). Cryo-EM volumes (pale cyan) around the ( c ) extracellular regions and ( d ) individual transmembrane helices of hFzd5. Except for the whole extracellular region (c right) which only shows disulfide cysteine resides with red colored secondary structures assigned by DSSP, all sidechains are displayed regardless of their quality or model placement.

Article Snippet: The following datasets were generated: Tsutsumi N Jude KM Gati C Garcia KC 2020 Structure of human Frizzled5 by fiducial-assisted cryo-EM Electron Microscopy Data Bank EMD-21927 Tsutsumi N Jude KM Gati C Garcia KC 2020 Structure of human Frizzled5 by fiducial-assisted cryo-EM RCSB Protein Data Bank 6WW2

Techniques: Cryo-EM Sample Prep, Generated