Journal: Immunology

Article Title: In vitro treatment of human transforming growth factor-? 1 -treated monocyte-derived dendritic cells with haptens can induce the phenotypic and functional changes similar to epidermal Langerhans cells in the initiation phase of allergic contact sensitivity reaction

doi: 10.1046/j.1365-2567.2000.00087.x

Figure Lengend Snippet: Monocyte-derived DCs treated with TGF-β1 induce E-cadherin and CLA. Peripheral blood CD14+ monocytes were cultured with GM-CSF and IL-4 (MoDCs) or with GM-CSF, IL-4 and TGF-β1 (TGF-β1+ DCs) for 6 days. The surface expression of several phenotypic markers for DCs or LCs were examined by flow cytometry. These are representative data from five different experiments that reproduced a similar staining pattern.

Article Snippet: We used the following monoclonal antibodies (mAbs) for immunostaining: fluorescein isothiocyanate (FITC)-anti-CLA, anti-CD40, anti-CD80, anti-CD86 antibodies, phycoerythrin (PE)-conjugated-anti-CD29, FITC or PE-conjugated isotype-matched mouse control antibodies [immunoglobulin G2a (IgG2a) and G2b; PharMingen, San Diego, CA], FITC- or PE-conjugated anti-HLA-DR antibody (Becton-Dickinson, San Jose, CA), FITC-conjugated anti-CD49e and -CD49f (Serotec Ltd, Oxford, UK), FITC-conjugated anti-CD49d, PE-conjugated anti-CD83 antibody (Immunotech, Marseilles, France), FITC-conjugated anti-CD54 antibody (Ancell, Bayport, MN), FITC-conjugated anti-CD44 antibody (Caltag Laboratories, Burlingame, CA), FITC-conjugated anti-HLA I-ABC antibody (Biosource, Camarillo, CA), PE-conjugated anti-CD1a antibody (Coulter, Hialeah, FL), monoclonal anti-human E-cadherin antibody (Takara Biomedicals, Tokyo, Japan), Lag (gift of Dr F. Furukawa, Hamamatsu University, Sizuoka, Japan) and isotype control antibody (IgG1) (Sigma).

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development *

doi: 10.1074/jbc.M113.469189

Figure Lengend Snippet: Expression and transport function of two SNP variants of hGLUT2. A, expression of hGLUT2 wild type (WT) and two SNP variants (P68L and T110I) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Plasma membrane location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to an extracellular epitope of hGLUT2 in nonpermeabilized cells. Nuclei are stained with DAPI (blue). Scale bar corresponds to 25 μm. B, Western blot analysis of membrane fractions from mhAT3F cells transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2 expression is revealed with an antibody to HA epitope. E-cadherin is used as a loading control of membrane fractions. C, membrane expression of WT, P68L, and T110I hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, P68L, or T110I hGLUT2 cRNA. Curves are fitted up using Michaelis-Menten nonlinear regression.

Article Snippet: Immunofluorescence Analyses Immunofluorescence on mhAT3F or MIN6 cells were performed as previously described ( 3 , 5 ) with anti-HA (sc-805; Santa Cruz), anti-hGLUT2 (directed against extracellular epitope of hGLUT2, MAB1414; R&D Systems), and anti-E-cadherin (TAK-M108, Takara) followed by cyanin-coupled secondary antibodies (715-165-150, 711-165-152, and 712-225-153; Jackson ImmunoResearch Laboratories Inc.).

Techniques: Expressing, Transfection, Construct, Staining, Western Blot, Injection

Journal: The Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development *

doi: 10.1074/jbc.M113.469189

Figure Lengend Snippet: Expression and transport function of four FBS-associated mutants of hGLUT2. A, expression of hGLUT2 wild type (WT) and four FBS-associated mutants (G20D, S242R, P417L, and W444R) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Subcellular location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to HA epitope in permeabilized cells. Nuclei are stained with DAPI (blue). Note that G20D hGLUT2 cannot be detected, and S242R hGLUT2 is not targeted at the plasma membrane. Scale bar corresponds to 25 μm. B, Western blot analysis of membrane fractions from mhAT3F cell transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2 expression is revealed with an antibody to HA epitope. E-cadherin is used as a loading control of membrane fractions. Note that G20D or S242R hGLUT2 cannot be detected in membrane fractions. C, membrane expression of WT, S242R, P417L, and W444R hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, noncorrected uptake of 2-DOG by Xenopus oocytes injected with water or injected with WT, S242R, P417L, or W444R hGLUT2 cRNA.

Article Snippet: Immunofluorescence Analyses Immunofluorescence on mhAT3F or MIN6 cells were performed as previously described ( 3 , 5 ) with anti-HA (sc-805; Santa Cruz), anti-hGLUT2 (directed against extracellular epitope of hGLUT2, MAB1414; R&D Systems), and anti-E-cadherin (TAK-M108, Takara) followed by cyanin-coupled secondary antibodies (715-165-150, 711-165-152, and 712-225-153; Jackson ImmunoResearch Laboratories Inc.).

Techniques: Expressing, Transfection, Construct, Staining, Western Blot, Injection

Journal: The Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development *

doi: 10.1074/jbc.M113.469189

Figure Lengend Snippet: Expression and transport functions of three engineered mutants of hGLUT2. A, membrane co-localization of wild type (WT), G20S, F295Y, and L368P hGLUT2 (red) and membrane marker E-cadherin (green) in infected mhAT3F cells analyzed by confocal microscopy. Nuclei are stained with DAPI (blue). Scale bar corresponds to 10 μm. B, similar migrating profiles of hGLUT2 WT and mutants (G20S, F295Y, and L368P) in membrane fractions of mhAT3F cells analyzed by Western blot. C, comparable membrane expression of WT, G20S, F295Y, and L368P hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, G20S, F295Y, and L368P hGLUT2. Curves are fitted up using Michaelis-Menten nonlinear regression.

Article Snippet: Immunofluorescence Analyses Immunofluorescence on mhAT3F or MIN6 cells were performed as previously described ( 3 , 5 ) with anti-HA (sc-805; Santa Cruz), anti-hGLUT2 (directed against extracellular epitope of hGLUT2, MAB1414; R&D Systems), and anti-E-cadherin (TAK-M108, Takara) followed by cyanin-coupled secondary antibodies (715-165-150, 711-165-152, and 712-225-153; Jackson ImmunoResearch Laboratories Inc.).

Techniques: Expressing, Marker, Infection, Confocal Microscopy, Staining, Western Blot, Injection

Journal: iScience

Article Title: N6-methyladenosine modification of B7-H3 mRNA promotes the development and progression of colorectal cancer

doi: 10.1016/j.isci.2024.108956

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were anti-B7-H3 (1:400; Proteintech, Cat No. 14453-1-AP), anti-ALDH (1:100; Abcepta, Cat No. AP1465c), anti-CD133 (1:500; Abcam, Cat No. ab27699), anti-MYC (1:500; Abcam, Cat No. ab18185), anti-NANOG (1:500; Novus, Cat No. NB100-58842), anti-SOX2 (1:300; Novus, Cat No. NB110-79875), anti-Ki67 (1:200; Proteintech, Cat No. 27309-1-AP), anti-Slug (1:1000; OriGene, Cat No. TA800167), anti-N-cadherin (1:5000; Proteintech, Cat No. 22018-1-AP) and anti-E-cadherin (1:2000; OriGene, Cat No. TA800692).

Techniques: Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis

doi: 10.3390/ijms25094939

Figure Lengend Snippet: Characteristics of the antibodies used.

Article Snippet: anti-α-ENaC , TA3250073 , polyclonal , WB 1:1000 IF 1:20 IP 2 µg , OriGene Technologies Inc. (Rockville, MD, USA).

Techniques:

Journal: Cellular and Molecular Life Sciences

Article Title: Downregulation of p53 drives autophagy during human trophoblast differentiation

doi: 10.1007/s00018-017-2695-6

Figure Lengend Snippet: Transcriptome analyses of BeWo cell differentiation. Immunofluorescence staining of E-cadherin in BeWo cells incubated either with vehicle control ( a , DMSO, 0.1%) or forskolin ( b , 20 µM). Stimulation with forskolin induced formation of multinucleated syncytia (outlined by dotted lines) after 48 h culture. Scale bar in ( b ) represents 100 µm. c qPCR analysis showed significantly upregulated mRNA expression of β subunit of human chorionic gonadotropin (CGB) in forskolin-treated BeWo cells after 48 h. Data are presented as mean ± SEM * p ≤ 0.05. d Bioinformatic pipeline applied to twofold regulated genes is depicted. e Top five positive (positive z -score) and negative (negative z -score) upstream regulators derived from ingenuity pathway analysis (IPA), sorted by adjusted p values given next to bars. f Heatmap of genes comprising the gene ontology (GO) term “autophagy” that is significantly enriched (adjusted p value = 7.9E−3) in a DAVID functional annotation analysis focused on GO biological processes. Data are from three independent experiments, using different cell passages

Article Snippet: Monoclonal anti-E-Cadherin antibody (Acris Antibodies GmbH, OriGene EU, Herford, Germany) was diluted 1:15 in antibody diluent (DAKO, Carpintera, CA, USA) and incubated on cells for 30 min. PBS washing steps were followed by incubation with secondary antibody, Alexa Fluor 555 goat anti-mouse (1:200; Invitrogen, Lifetechnologies, Carlsbad, CA, USA) for 30 min.

Techniques: Cell Differentiation, Immunofluorescence, Staining, Incubation, Expressing, Derivative Assay, Functional Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Downregulation of p53 drives autophagy during human trophoblast differentiation

doi: 10.1007/s00018-017-2695-6

Figure Lengend Snippet: Effect of forskolin on p53 and autophagy in non-fusing JAR trophoblasts. Immunofluorescence staining of E-cadherin in JAR cells incubated either with vehicle control ( a , DMSO, 0.1%) or forskolin ( b , 20 µM) did not show formation of multinucleated syncytia after 48 h culture. qPCR analysis of p53 ( c ) and LC3B ( d ) mRNA as well as immunoblotting ( e ) with subsequent band densitometry for p53 ( f ), LC3B-I, and LC3B-II ( g – i ) protein levels did not show significant differences between forskolin (20 µM) treated JAR cells and vehicle control (DMSO, 0.1%) after 48 h. Scale bar in b represents 100 µm. Data in c , d and f – i are presented as mean ± SEM from three independent experiments, using different cell passages

Article Snippet: Monoclonal anti-E-Cadherin antibody (Acris Antibodies GmbH, OriGene EU, Herford, Germany) was diluted 1:15 in antibody diluent (DAKO, Carpintera, CA, USA) and incubated on cells for 30 min. PBS washing steps were followed by incubation with secondary antibody, Alexa Fluor 555 goat anti-mouse (1:200; Invitrogen, Lifetechnologies, Carlsbad, CA, USA) for 30 min.

Techniques: Immunofluorescence, Staining, Incubation, Western Blot

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A and B) Hyphae of the indicated strains of C. albicans were incubated for 45 min with human umbilical vein endothelial cells (A) or the FaDu oral epithelial cell line (B), after which the number of endocytosed and cell-associated (endocytosed plus adherent) organisms was determined by a differential fluorescence assay. The data are expressed as a percentage of the results with wild-type organisms, and are the mean ± SD of three experiments, each performed in triplicate. *, p < 0.001 compared to the wild-type strain and the als3 Δ/ als3 Δ:: ALS3 -complemented strain. (C and D) Binding of surface proteins from endothelial cells (C) and FaDu oral epithelial cells (D) to hyphae of the indicated strains of C. albicans . Biotinylated endothelial or epithelial cell membrane proteins that bound to C. albicans were eluted from the hyphae with urea and separated by SDS-PAGE. (C) The same immunoblot of endothelial cell surface proteins was developed with an anti-biotin monoclonal antibody (top) and an anti-N-cadherin monoclonal antibody (bottom). (D) An immunoblot of oral epithelial cell surface proteins was developed with an anti-biotin monoclonal antibody (top) and an anti-E-cadherin monoclonal antibody (bottom). Arrows in (C) and (D) indicate the bands that correspond to N-cadherin and E-cadherin, respectively.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Fluorescence, Binding Assay, Membrane, SDS Page, Western Blot

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A) Number of hyphae of the indicated clinical isolates of C. albicans that were endocytosed by and cell-associated with the FaDu oral epithelial cell line after a 45-min incubation. The data are expressed as a percentage of the results with strain DAY185, and are the mean ± SD of three experiments, each performed in triplicate. *, p < 0.05 compared to strain DAY185. (B and C) Immunoblot of biotinylated FaDu cell surface proteins eluted from the indicated strains of C. albicans. The same blot was probed with antibodies against biotin (B) or E-cadherin (C). (D) Flow cytometric detection of Als3 expression on the surface of C. albicans hyphae. Hyphae of the indicated strains were stained using indirect immunofluorescence with either a polyclonal rabbit anti-Als3 antiserum (solid lines) or purified rabbit IgG (broken lines) and then analyzed using flow cytometry.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Western Blot, Expressing, Staining, Immunofluorescence, Purification, Flow Cytometry

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: Confocal micrographs of uninfected endothelial cells (A–C), or endothelial cells infected with the wild-type strain (D–G), the als3 Δ/ als3 Δ null mutant (H–K), or the als3 Δ/ als3 Δ:: ALS3 -complemented strain (L–O). The cells were stained for N-cadherin (A), (D), (H), and (L), actin microfilaments (B), (E), (I), and (M), and C. albicans (F), (J), and (N). The merged images are shown in (C), (G), (K), and (O). Arrows indicate the accumulation of N-cadherin and microfilaments around the organisms. Bar in (O) indicates 10 μm.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Infection, Mutagenesis, Staining

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: Confocal micrographs of uninfected FaDu oral epithelial cells (A–C) or epithelial cells infected with the wild-type strain (D–G), the als3 Δ/ als3 Δ null mutant (H–K), or the als3 Δ/ als3 Δ:: ALS3 -complemented strain (L–O). The cells were stained for E-cadherin (A), (D), (H), and (L), microfilaments (B), (E), (I), and (M), and C. albicans (F), (J), and (N). The merged images are shown in (C), (G), (K), and (O). Arrows indicate the accumulation of E-cadherin and microfilaments around the organisms. Bar in (O) indicates 10 μm.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Infection, Mutagenesis, Staining

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A–D) Latex beads were coated with BSA, rAls1-N, or rAls3-N. They were incubated for 45 min with endothelial cells (A), FaDu oral epithelial cells (B), CHO cells expressing human N-cadherin (C), or CHO cells expressing human E-cadherin (D), after which the number of endocytosed and cell-associated beads was determined. (E) The interactions of BSA- and rAls3-N–coated beads with control CHO cells expressing no human cadherins (Control) were compared with those of CHO cells expressing human E-cadherin (E-cadherin) by the same method as in (A–D). Data are expressed as a percentage of the results with beads coated with BSA and are the mean ± SD of three or four experiments, each performed in triplicate. *, p ≤ 0.005 compared to beads coated with BSA; † p < 0.05 compared to beads coated with BSA; § p < 0.005 compared to beads coated with rAls1-N; and ‡ p ≤ 0.01 compared to control CHO cells.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Expressing, Control

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: The most distal (N1) and adjacent (N2) N-terminal domains of Als1, Als3, E-cadherin, and N-cadherin are shown. The predicted structures of the N-terminal regions of Als1 and Als3 were extracted from their larger models, and structures for E-cadherin and N-cadherin were obtained from the Protein Data Bank . (A) Comparative secondary structures. Yellow color indicates β-sheet propensity; red, α-helical propensity; aqua, extended or turn structures. Note that the Als1 model encompasses 187 amino acids (residues 3–190) in an equivalent 3-D space as that of Als3, which includes 279 amino acids (residues 20–299). Such a comparison illustrates the overall similarities in conformation among these polypeptide domains, with a more compact configuration of Als3. (B) Comparative hydrophobic surfaces. Surface hydrophobicity is illustrated in color upon the solvent-accessible surface areas of respective molecules. For clarity, only the hydrophobic surface is colored; the ghost structures (gray) represent the polar surface. In hydrophobic areas, brown indicates the most hydrophobic regions and blue indicates the least hydrophobic regions. (C) Comparative electrostatic surfaces. Negatively charged electrostatic surface is illustrated in color upon the solvent accessible surface areas of respective molecules. For clarity, only the negatively charged surface is colored; the ghost structures (gray) represent the small area on each molecule bearing a positive charge. In negatively charged areas blue indicates the strongest negative surface potential and yellow indicates the weakest negative charge potential.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Comparison, Solvent

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: The interactions of the N-terminal regions of Als1 and Als3 with the N-terminal regions of N-cadherin and E-cadherin, as well as N-cadherin and E-cadherin self-association were modeled as outlined in Materials and Methods. The N-terminal domains of Als1 and Als3 are shown in their energy-minimized conformations; docking results using alternate conformations yielded equivalent results (unpublished data). (A) The solvent-accessible surface areas of the proteins are shown, and the β barrel domains are numbered sequentially from the N-terminus. In models of the docking of an Als protein to a cadherin (left and middle columns), the Als protein is blue and the cadherin is gold. In models of cadherin self-association, one cadherin molecule is blue and the other cadherin is gold. (B) Interactions of Als1 or Als3 with E-cadherin or N-cadherin are presented to illustrate their predicted docking characteristics. In the top two rows, the indicated cadherin electrostatic contact surfaces are superimposed upon the backbone structures of Als1 or Als3. Coloration schema of the Als secondary structure: gold, β sheet; blue, extended/turn. Coloration schema of the cadherin surface: blue, most negative; red, most positive. In the bottom two rows, the contact footprints of the indicated cadherins (dark blue) are superimposed upon the solvent-accessible surface areas of Als1 or Als3 (aqua) to indicate the predicted docking positions of the protein pairs. In all cases, note that the ectodomain N2 of Als3 contributes to the interactions with both cadherins, while the corresponding ectodomain N2 of Als1 does not participate in interactions with either cadherin.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Solvent