Journal: bioRxiv

Article Title: Lgr5+ telocytes are a signaling hub at the intestinal villus tip

doi: 10.1101/850909

Figure Lengend Snippet: VTTs implement a spatial switch from canonical to non-canonical Wnt signaling. a) Volcano plot demonstrating differential gene expression between VTTs and crypt telocytes. Black dots have qval<0.2 and expression fold change larger than 2 or smaller than ½. Wnt5a and Lgr5 are marked in red. b) Spatial shift in the stroma from the expression of canonical Wnt2b (red dots, marked by red arrows) to non-canonical Wnt5a (green dots, marked by green arrows). Scale bar – 20 µm. c) Axin2 (red dots) is expressed broadly along the villus axis and repressed at the villus tip. Fos (green dots) is highly expressed in villus tip enterocytes. scale bar – 20 µm. d) E-Cadherin protein (gray), encoded by Cdh1 gene, is induced in villus tip enterocytes. Large blobs in B-D are autofluorescent signals originating in immune cells.

Article Snippet: For E-cadherin immunofluorescence staining FITC anti E-Cadherin (612131 BD Biosciences) was used at dilution of 1:100.

Techniques: Expressing

Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Multicellular Human Primary Endometrial Organoids

doi: 10.3791/60384

Figure Lengend Snippet: Table of Materials

Article Snippet: Purified mouse anti-E-cadherin antibody , BD Biociences , 610181 , Clone 36.

Techniques: Saline, Electron Microscopy, Staining, Modification, Formulation, Purification, Recombinant

Journal: Epigenetics

Article Title: Targeted disruption of the histone lysine 79 methyltransferase Dot1L in nephron progenitors causes congenital renal dysplasia

doi: 10.1080/15592294.2020.1861168

Figure Lengend Snippet: Antibodies used in the immunostaining assays

Article Snippet: Primary Antibody Cat# Source Working dilution Mouse anti-E-cadherin 610,181 BD Biosciences 1:100 Rabbit anti Six2 11,562-1-AP Proteintech group 1:200 LTA FL-1321 Vector Laboratories 1:100 Rabbit anti-Pax2 716,000 Invitrogen 1:100 Goat anti-AQP-2 9 C17) sc-9882 Santa Cruz 1:100 Rabbit anti-WT1 (C-19) sc-192 Santa Cruz 1:100 Mouse anti-Lhx1 4F2-c DHB 1:100 Rabbit anti-WT1 Ab15249 Abcam 1:100 Rabbit anti-PDGFR β C82A3 Cell Signalling 1:400 Rabbit anti-Pecam1 (M20) Sc-1506 SantaCruz 1:600 Rabbit anti-MHC Ab124679 Abcam 1:200 Rabbit anti-renin Gomez lab* 1:500 Mouse anti-NCAM C9672 Sigma 1:200 Rabbit anti-angiotensinogen #28,101 IBL 1:200 Rabbit anti-NKCC2 PA5-80,003 Invitrogen 1:200 Rabbit anti-N1ICD 4147S Cell Signalling 1:200 Rabbit anti-Notch2 5732S Cell Signalling 1:200 Rabbit anti-Dot1L Ab64077 Abcam 1:1000 Open in a separate window *Gomez RA et al. Am J Physiol Heart Circ Physiol 296: H1255-H1262, 2009.

Techniques: Immunostaining, Plasmid Preparation

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: Cell–cell contacts are maintained in cells depleted of E-cadherin. Immunofluorescence staining of MDCK cells fixed 2 d posttransfection with E-cadherin pSEcad or pSLuc shRNAs. YFP expression is a transfection marker. (A) TJ marker proteins ZO-1, Pals1, Par3, and the transmembrane protein Claudin1 localize to cell–cell contacts in control and knockdown cells. (B) Adherens junction proteins E-cadherin, β-catenin, α-catenin, and actin localization at cell–cell contacts in both control and knockdown cells.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Immunofluorescence, Staining, Expressing, Transfection, Marker

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: β-catenin is in excess of E-cadherin in MDCK cells. (A) Western blot analysis of whole cell lysates 2 d posttransfection with pSLuc or pSEcad vectors. Lysates were blotted for E-cadherin, β-catenin, and α-catenin to determine relative protein abundances. (B) β-catenin is in excess of E-cadherin in MDCK cells. TX-100 lysates from untransfected cells were analyzed by immunoblot after serial E-cadherin immunodepletion. (C) MDCK cells were lysed in a TX-100 lysis buffer, and samples of the insoluble (pellet) and soluble (sup) fractions were analyzed by immunoblot. The fraction of β-catenin in the insoluble pool does not change in cells after E-cadherin depletion. Soluble lysate fractions were immunoprecipitated with β-catenin antibody or mouse nonimmune antibody (NIIgG) as a control. Samples were blotted for β-catenin. (D) Immunoblot of cell lysates from control (pSLuc) or pS-Ecad–transfected cells. Lysates were blotted for E-cadherin and Pan-cadherin. β-tubulin is a loading control. (E) Whole cell lysates of control (pSLuc) and pSEcad cells blotted for Cadherin-6, E-cadherin, and β-tubulin.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Western Blot, Lysis, Immunoprecipitation, Transfection

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: Simultaneous suppression of Cadherin-6 and E-cadherin causes mislocalization of β-catenin, yet TJs are not disrupted. (A) Immunofluorescence of Cadherin-6 in MDCK cells fixed 2 d after transfection with E-cadherin (pSEcad) or pSLuc shRNA vectors. YFP is the transfection marker. (B) Immunoblot analysis of cell lysates from MDCK cells transfected with pSLuc or pSEcad, Cadherin-6 shRNA vector (pSCad6), or both pSEcad plus pSCad6. (C) Immunofluorescence images of MDCK cells depleted of Cadherin-6 or of both E-cadherin and Cadherin-6. Cells were fixed and stained with anti-Cadherin-6 or anti-E-cadherin to confirm cadherin suppression in transfected cells. Cellular localization of ZO-1, β-catenin, α-catenin, and actin is shown for both Cadherin-6 and double knockdown conditions. Transfected cells express YFP.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Immunofluorescence, Transfection, shRNA, Marker, Western Blot, Plasmid Preparation, Staining

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: Suppression of α-catenin disrupts confluent MDCK cells. (A) Immunoblot of cell lysates 2 d posttransfection with control, α-catenin and/or E-cadherin shRNA vectors. (B) DIC image of MDCK cells at low confluence (top) or high confluence (bottom) transfected with either control or α-catenin shRNA vectors. (C) Immunofluorescence images of α-catenin–depleted MDCK cells stained for α-catenin, ZO-1, β-catenin, and E-cadherin. YFP was a transfection marker. (D) Recompiled confocal sections of α-catenin–depleted cells grown on filters. Cells were fixed and stained for the apical marker gp135 (green). mRFP was used as a transfection marker (red). The right-hand panels are cross-sections of the same image.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Western Blot, shRNA, Transfection, Immunofluorescence, Staining, Marker

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: TJ biogenesis is disrupted in cells depleted of E-cadherin. E-cadherin knockdown cells fail to polarize properly after calcium switch. (A) Immunofluorescence of MDCK cells 2 d after transfection with control shRNA and YFP. (B) E-cadherin–depleted cells fixed and stained 2 d after transfection. Cells were stained for ZO-1 and β-catenin. YFP marks transfected cells. (C) Average TER after calcium switch for control (■) and knockdown cells (▴). (D) Trypsinized and replated cells were analyzed by immunofluorescence for TJ formation. Cells were fixed 7 h after replating and stained for E-cadherin and ZO-1.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Immunofluorescence, Transfection, shRNA, Staining

Journal:

Article Title: Depletion of E-Cadherin Disrupts Establishment but Not Maintenance of Cell Junctions in Madin-Darby Canine Kidney Epithelial Cells

doi: 10.1091/mbc.E06-05-0471

Figure Lengend Snippet: E-cadherin knockdown is partially rescued by E-cadherin–α-catenin fusion protein. (A) Live cell time-lapse imaging of YFP-ZO-1 MDCK cells after calcium switch. Cells were transfected with vectors for control or E-cadherin shRNAs or the E-cadherin–α-catenin fusion protein (Ecat). mRFP marks transfected cells. Videos are available in Supplementary Material. (B) Quantification of ZO-1 length as a measure of TJ recovery (pixels per cell). Average lengths are recorded up to 120 min after calcium switch for control cells (■) E-cadherin depleted cells (▴) and depleted cells plus an E-cadherin–α-catenin fusion protein (▾). (C) Immunoblot of cell lysates from YFP-ZO-1 cells transfected with pSLuc, pSEcadB, or pSEcadB vectors plus the E-cadherin–α-catenin fusion protein.

Article Snippet: Primary antibodies were rabbit anti-β-tubulin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZO-1 (1:1000; Zymed Laboratories), mouse anti-E-cadherin and β-catenin (1:1000; BD Biosciences), rabbit anti-α-catenin (1:2000; Sigma-Aldrich), rabbit anti-Pals1 (1:1000) and mouse anti-Ran (1:5000; BD Transduction Laboratories, Lexington, KY), and mouse anti-Rac (1:1000; Upstate Biotechnology).

Techniques: Imaging, Transfection, Western Blot