Journal:

Article Title: Herpes Simplex Virus Type 2 Induces Rapid Cell Death and Functional Impairment of Murine Dendritic Cells In Vitro

doi: 10.1128/JVI.77.20.11139-11149.2003

Figure Lengend Snippet: Subpopulation of HSV-2-infected BMDC exhibit DNA breaks shortly after infection. BMDC were infected with wt HSV-2 at an MOI of 4 or left untreated, incubated for up to 12 h p.i., and then double stained for HSV ICP8 (anti-ICP8 MAb 383 plus FITC-conjugated goat anti-mouse MAb) and for apoptosis (biotin-labeled dCTP followed by avidin-Texas Red). Shown are immunofluorescence microscope images of the same optical section of a population of HSV-2-infected DC collected 2 h p.i. to demonstrate staining for ICP8 (green) (A), nick end-labeled DNA breaks (TUNEL) (red) (B), and a double-labeled, HSV-2-infected, apoptotic BMDC (yellow). Arrow in panels A, B, and C indicates positive staining of an apoptotic, HSV-2-infected BMDC. Included were double-stained uninfected BMDC as a negative control (D) and DNase-treated uninfected DC stained with biotin-dCTP-avidin-Texas red as a positive control (E) for the TUNEL assay. Magnification, ×40.

Article Snippet: Cytospin slides were prepared and fixed as described above, blocked with 20% goat serum (Jackson ImmunoResearch), and then stained with antibodies against HSV ICP4 (M612446; BioClone), ICP8 (383; a kind gift from David Knipe, Harvard Medical School, Boston, Mass.), or HSV-2 glycoprotein D (gD) (910; Biodesign International, Saco, Maine), followed by FITC-conjugated goat anti-mouse MAb (Jackson ImmunoResearch).

Techniques: Infection, Incubation, Staining, Labeling, Avidin-Biotin Assay, Immunofluorescence, Microscopy, TUNEL Assay, Negative Control, Positive Control

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 2. Amino acids 1-63 of AE1-4 can direct a cytoplasmic tailless mutant of the Fc receptor to the basolateral membrane of MDCK cells. Confluent MDCK cells stably expressing Fc– (A), Fc1-63 (B), Fc1-63Y44A (C), Fc1-63Y47A (D) or Fc1-63Y44A,Y47A (E) were fixed and incubated with the rat monoclonal antibody specific for the Fc receptor and phalloidin-FITC. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine, and the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center (B, C and D) or near the apical surface (A and E) of the cells. Regions that are yellow indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image of the transfected cells. The bar in each xy image is 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Membrane, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Transfection

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 3. Amino acids 1-37 and 38-63 of AE1-4 target a cytoplasmic tailless Fc receptor to distinct intracellular membrane compartments. MDCK cells stably expressing Fc1-37 (A-C) or Fc38-63 (D-F) were fixed and incubated with rat monoclonal antibodies specific for the Fc receptor (A and D), a mouse monoclonal antibody specific for the mannose 6-phosphate receptor (B) or a rabbit polyclonal antibody specific for furin (E). The cells were washed and incubated with donkey anti-rat IgG conjugated to lissamine (A and D), goat anti- mouse IgG conjugated to FITC (B) or donkey anti-rabbit IgG conjugated to FITC (E). The localization of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The merged images illustrate significant overlap in the distribution of Fc 1-37 and the mannose 6-phosphate receptor (C) and Fc38-63 and furin (F). Bar in A and D, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Membrane, Stable Transfection, Expressing, Incubation, Bioprocessing, Labeling, Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 4. Fc1-37 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of the mannose 6-P receptor. MDCK cells stably expressing Fc1-37 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a mouse monoclonal directed against the mannose 6-phosphate receptor (M-6-P). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and goat anti-mouse IgG conjugated to FITC. Following washing, the localization of Fc1-37 (A,B) and the mannose 6-phosphate receptor (C,D) was visualized on a Zeiss LSM 510 laser-scanning microscope. The merged images showing the overlap of Fc1-37 and the mannose 6-phosphate receptor are shown in E and F. The arrows in A indicate the Fc1-37 chimeras that still reside on the cell surface following a 15 minute incubation at 37°C. Bar (A,B), 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Clinical Proteomics, Membrane, Stable Transfection, Expressing, Incubation, Laser-Scanning Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 5. Fc38-63 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of furin. MDCK cells stably expressing Fc38-63 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a rabbit polyclonal directed against furin. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and donkey anti-rabbit IgG conjugated to FITC. Following washing, the localization of Fc38-63 (A,B) and furin (C,D) was visualized on a Zeiss LSM 510 laser scanning microscope. The merged images showing the overlap of Fc38-63 and furin are shown in E and F. Bar (A,B), 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Clinical Proteomics, Membrane, Stable Transfection, Expressing, Incubation, Laser-Scanning Microscopy

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 6. The surface distribution and steady-state localization of wild- type and mutant Fc38-63 constructs in polarized MDCK cells. Polarized MDCK cells stably expressing Fc38-63 (A,B) or Fc38- 63Y47A (C) or transiently expressing Fc38-63Y47L (D) or Fc38- 63L50A (E) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B-E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-E) and phalloidin conjugated to FITC (B-E). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center of the cells. Regions that are yellow in B-E indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Construct, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Membrane

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 7. The surface distribution and steady-state localization of wild- type and mutant Fc1-37 constructs in polarized MDCK cells. Polarized MDCK cells transiently (A,C) or stably (B) expressing Fc1- 37 (A and B) or Fc1-37S25A (C) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B,C). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-C) and phalloidin conjugated to FITC (B and C). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is either near the center (A,B) or near the apical surface (C) of the cells. The surface population of Fc1-37 could be detected in a longer exposure of the image in B. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Mutagenesis, Construct, Stable Transfection, Expressing, Incubation, Labeling, Microscopy, Membrane

Journal: Journal of cell science

Article Title: Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

doi: 10.1242/jcs.00260

Figure Lengend Snippet: Fig. 8. Fc1-63 and Fc38-63Y47A colocalize with phalloidin-stained stress fibers in subconfluent MDCK cells. Subconfluent MDCK cells stably expressing Fc1-63 (A-C) or Fc38-63Y47A (D-F) were fixed, permeabilized and incubated with the Fc-receptor-specific antibody (A,D) and phalloidin conjugated to FITC (B,E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine. Following washing, the localization of fluorescently labeled polypeptides was visualized on a Zeiss Axiophot microscope. The merged images showing the overlap of these chimeras with phalloidin-stained microfilaments are shown in C and F. Bars, 10 µm.

Article Snippet: The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch).

Techniques: Staining, Stable Transfection, Expressing, Incubation, Labeling, Microscopy

Journal: The European respiratory journal

Article Title: Classical protein kinase C and its hypoxic stimulus-induced translocation in the cat and rat carotid body.

doi: 10.1034/j.1399-3003.2000.016003459.x

Figure Lengend Snippet: Fig. 1. ± Confocal fluorescence microscopy of double immunolabelling of a section of a normoxic cat carotid body tissue stained with mouse anti- protein kinase C (PKC)c and rabbit anti-tyrosine hydroxylase (TH). The localization of PKCc is visualized in green with fluorescein isothiocyanate labelled antimouse immunoglobulin G (IgG) (A), whereas that of TH in red with Texas red-labelled antirabbit IgG (B). An overlapping image is shown in C; the yellow spots indicate colocalization of the two fluorescent probes. The near-perfect colocalization identifies the PKCc as being in the chemoreceptor cell. (Internal scale bars=10 mm.)

Article Snippet: For immunofluorescence staining, sections were incubated with fluorescein isothiocyanate (FITC)-labelled antimouse immunoglobulin G (IgG; dilution 1:200; Jackson Immunoresearch West Grove, PA, USA) for 1 h. To confirm the chemoreceptor cell localization of the PKC antigens, other sections were double-labelled by simultaneous incubation with a rabbit polyclonal antibody directed against rat tyrosine hydroxylase (TH; dilution 1:500; Chemicon International, Inc., Temecula, CA, USA) and visualization with Texas red-labelled antirabbit IgG (dilution 1:200; Jackson Immunoresearch).

Techniques: Fluorescence, Microscopy, Staining

Journal: Eukaryotic Cell

Article Title: Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

doi: 10.1128/ec.00134-07

Figure Lengend Snippet: FIG. 1. Levels of polytopic membrane proteins under conditions of SRP knockdown. (A) Effect on TbNT8.1-GFP. (A1) Membrane and soluble fractions were prepared as described in Materials and Methods from uninduced (Tet) and SRP54-silenced (Tet) cells on the third day after induction and were subjected to Western blot analysis with anti-GFP and anti-EP antibodies. Anti-hnRNPD0 antibodies were used to control for the quality of fractionation. The positions of fused as well as cleaved GFP are indicated. (A2) Cells were fixed with 4% formaldehyde for 25 min and visualized by confocal microscopy. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bar, 5 m. (B) Effect on VHppase. (B1) Western blot analysis of lysates from uninduced cells and SRP54-silenced cells on the third day after induction with anti-VHppase antibodies. hnRNPD0 was used to control for equal loading. (B2) Cells were fixed with 4% formaldehyde for 25 min, incubated with anti-VHppase antibodies, and detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Scale bar, 5 m. (C) Effect on Tb29 proteins. Western blot analysis of uninduced cells and SRP68-silenced cells was performed on the third day after induction with anti-Tb29 antibodies. Tb29-2 is the Tb29 protein containing the TM domain; Tb29-1 is the Tb29 protein carrying an SP. hnRNPD0 reactivity with antibodies was used to control for equal loading. (D) Effect of inhibition of proteolysis by the proteasome or lysosome on VHppase. Uninduced and SRP68-silenced cells on the third day after induction were treated with 5 M lactacystin or 0.25 M bafilomycin for 4 h. Proteins were prepared as described in Materials and Methods from lactacystin (a)- or bafilomycin (b)-treated uninduced or SRP68-silenced cells and were subjected to Western blot analysis with anti-VHppase antibodies. hnRNPD0 reactivity with antibodies was used to control for equal loading. (E) Immunoprecipitation of in vivo-labeled VHppase protein. Uninduced and induced cells on the third day after tetracycline induction were labeled with L-[35S]Met-Cys for 3 h. Extracts were prepared, and immunoprecipitation was performed, as described in Materials and Methods. The total-cell extract (Total) and the immunoprecipitated products (IP) were analyzed on a 10% SDS-polyacrylamide gel. The sizes of marker proteins are given on the left. The specific immuno- precipitated product is indicated by an arrow.

Article Snippet: Cells were first incubated with PBS containing 10% fetal calf serum at room temperature for 30 min and then incubated with the primary antibodies (anti-VH ppase [1:500] or anti-BiP [1:200]) in PBS–0.1% Nonidet P-40 for 1 h. After a wash with PBS, the cells were reacted with anti-IgG conjugated to fluorescein isothiocyanate (FITC; Jackson ImmunoResearch).

Techniques: Membrane, Knockdown, Western Blot, Control, Fractionation, Confocal Microscopy, Incubation, Inhibition, Immunoprecipitation, In Vivo, Labeling, Marker

Journal: Eukaryotic Cell

Article Title: Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

doi: 10.1128/ec.00134-07

Figure Lengend Snippet: FIG. 3. (A1) Level of 7SL RNA in cells carrying the SP-GFP-TM reporter. RNA was prepared from wild-type (WT) cells and from cells carrying the 7SL RNA A2 or A4 mutant on pX-hygro; all cells also carried SP-GFP-TM on the pX-neo plasmid. RNA was separated on a denaturing gel and subjected to Northern blot analysis with an antisense oligonucleotide to 7SL RNA. The positions of 7SL I and 7SL II are indicated. (A2) Biochemical fractionation of SP-GFP-TM. Membrane and soluble fractions were prepared from 109 cells, and aliquots were separated on 12% SDS-polyacrylamide gels and subjected to Western blot analysis with anti-GFP antibodies. The identities of the cell lines used for preparing the samples are indicated. (A3) Confocal microscopy of cells expressing SP-GFP-TM. Cells were treated with FM-4-64 for 40 min, immobilized in agarose, and visualized by confocal microscopy. The images presented, from left to right, are as follows: cells treated with FM-4-64 for 40 min, GFP fluorescence, merging of the FM-4-64 and GFP images, and merging of the FM4-64, GFP, and DIC images. The cell types are given on the left. L, lysosome; E, endosome. (B) The level of BiP does not change in cells carrying 7SL RNA mutants. (B1) Proteins (20 g) from WT cells and cells carrying the A4 or A2 mutation were subjected to Western blot analysis with anti-BiP antibodies. Reactivity with anti-hnRNPD0 antibodies was used to control for equal loading. (B2) Cells were fixed with 4% (vol/vol) formaldehyde for 25 min and incubated with anti-BiP antibodies, which were detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Nuclei were stained with DAPI. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bars, 5 m.

Article Snippet: Cells were first incubated with PBS containing 10% fetal calf serum at room temperature for 30 min and then incubated with the primary antibodies (anti-VH ppase [1:500] or anti-BiP [1:200]) in PBS–0.1% Nonidet P-40 for 1 h. After a wash with PBS, the cells were reacted with anti-IgG conjugated to fluorescein isothiocyanate (FITC; Jackson ImmunoResearch).

Techniques: Mutagenesis, Plasmid Preparation, Northern Blot, Fractionation, Membrane, Western Blot, Confocal Microscopy, Expressing, Control, Incubation, Staining

Journal: Eukaryotic Cell

Article Title: Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

doi: 10.1128/ec.00134-07

Figure Lengend Snippet: FIG. 4. Localization of polytopic membrane proteins in wild-type (WT) cells and cells carrying 7SL RNA mutants. (A1) Western blot analysis of soluble and membrane-bound GT3-GFP. Membrane and soluble fractions were prepared from 109 cells, and aliquots were subjected to Western blot analysis with anti-GFP antibodies. Ponceau staining or reactivity against anti-hnRNPD0 antibodies was used to control for equal loading of the membrane or soluble fraction, respectively. The positions of GT3-GFP and prGFP are indicated. (A2) GFP fluorescence in WT cells and 7SL RNA mutants carrying pX-GT3-GFP. Cells were fixed in agarose and treated with FM-4-64 for 40 min. The images are as described in the legend to Fig. 3A3. L, lysosome; E, endosome; FP, flagellar pocket. (B) Level and localization of VHppase in WT cells and cells carrying 7SL RNA mutants. (B1) Proteins (20 g) from WT cells and cells carrying the A4 or A2 mutation were subjected to Western blot analysis with anti-VHppase antibodies. Reactivity against anti-hnRNPD0 antibodies was used to control for equal loading. (B2) Cells were fixed with 4% (vol/vol) formaldehyde for 25 min and incubated with anti-VHppase antibodies, which were detected by an FITC-conjugated secondary antibody. Cells were visualized by confocal microscopy. Nuclei were stained with DAPI. DIC, differential interference contrast; IFA, immunofluorescence assay. Scale bars, 5 m.

Article Snippet: Cells were first incubated with PBS containing 10% fetal calf serum at room temperature for 30 min and then incubated with the primary antibodies (anti-VH ppase [1:500] or anti-BiP [1:200]) in PBS–0.1% Nonidet P-40 for 1 h. After a wash with PBS, the cells were reacted with anti-IgG conjugated to fluorescein isothiocyanate (FITC; Jackson ImmunoResearch).

Techniques: Membrane, Western Blot, Staining, Control, Mutagenesis, Incubation, Confocal Microscopy

Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet: CD226 expression is higher on NK cells in Il22 −/− mice (A) Intravenous model of 4T1-luciferase + (4T1-Luc) metastasis. (B) Representative IVIS images and average radiance from one experiment of two (n = 5). (C) Ex vivo imaging of the lung by IVIS. Linear regression analysis of clonogenic colonies vs. average radiance. (D) Representative dot plots and gating strategy of IFNγ-producing cells in the lungs. Numbers represent the frequency of the parent gate. Frequency of IFNγ + cells in the lung and composition by cell type including CD8 + and CD4 + T cells, NKT cells, and NK cells. (E) Representative dot plots and frequency of IFNγ + NK cells (n = 9 and 10). Data from two independent experiments were pooled. (F) Representative fluorescent microscopy images of lungs from 4T1-injected mice. NKp46 FITC signal is depicted in red, CD8 AF555 in yellow, CD155 PE in green, and Hoechst DNA staining in blue. Intralesional CD155 PE MFI per lesion in WT and Il22 −/− mice. Chip cytometry spatial distribution of NKp46 + (red), CD8 + (green), and CD155 + (gray) cells in the microscopy samples. Frequency of CD8 and NKp46 cells per lesion from the previous graph. Data presented as means ± SEM; p values <0.05 were considered significant by paired t test of one chip of 2 for each condition (n = 3 and 4). (G) Gating strategy and representative plots of CD226 staining on CD8 + T, CD4 + T, NKT, and NK cells. Isotype control is in black. Frequency of CD226 + cells in the lungs (n = 6). Data of one experiment of two. Data presented as means ± SEM; p values <0.05 were considered significant by the Mann-Whitney U test or multiple unpaired t tests with Holm-Sidak correction. See also Figure S6 .

Article Snippet: The following day, FITC conjugated donkey anti-goat antibody (Jackson ImmunoResearch) was applied at a concentration of 10 μg/mL for 2 h at room temperature.

Techniques: Expressing, Luciferase, Ex Vivo, Imaging, Microscopy, Injection, Staining, Chip Cytometry, Control, MANN-WHITNEY

Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet:

Article Snippet: The following day, FITC conjugated donkey anti-goat antibody (Jackson ImmunoResearch) was applied at a concentration of 10 μg/mL for 2 h at room temperature.

Techniques: Purification, Control, Virus, Recombinant, Red Blood Cell Lysis, Transfection, Flow Cytometry, DNA Extraction, Gel Extraction, Plasmid Preparation, cDNA Synthesis, Cell Differentiation, Staining, Cell Isolation, Conjugation Assay, Sequencing, CRISPR, Retroviral, Software