goat anti rabbit igg Thermo Fisher Search Results


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  • 98
    Thermo Fisher secondary antibody
    Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 10102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg h l cross adsorbed secondary antibody
    Loss of VCP induces EMT A. 72 hours after transfection of A549, H358 or immortalized human bronchiolar HPLD-1 cells with siRNA against VCP or non-targeting cell lysates were prepared and western blot analysis of EMT markers Vimentin and E-cadherin was performed. B. Fluorescence staining for E-cadherin and Vimentin in A549 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using <t>Alexa</t> Fluor 488 goat anti-rabbit <t>IgG</t> (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. C. A549 cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.
    Goat Anti Rabbit Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 15592 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 goat anti rabbit igg
    Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with <t>Alexa</t> <t>Fluor</t> 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p
    Alexa Fluor 488 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8093 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg
    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells <t>(RBCs)</t> to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. <t>IgG1,</t> immunoglobulin G1.
    Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 conjugated goat anti rabbit igg
    Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with <t>Alexa</t> Fluor 488-conjugated anti-rabbit
    Alexa Fluor 488 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 goat anti rabbit igg
    Drosophila lacking dLRRK kinase activity has normal development of DA neuron . Brain dissected from wild-type flies (a), e03680/+ flies (b) and e03680/e03680 flies (c) aged 20 days were immunostained with anti- Drosophila TH antibody followed by an <t>Alexa</t> Fluor 594-labeled secondary antibody to indentify DA neurons. Representative pictures shown were collected by confocal microscopy. Dopaminergic neurons in six brain regions, including PAL, PPM1/2, PPM3, PPL1, PPL2, and VUM, were quantified and no significant difference was found among different fly lines (d). Localization of Drosophila DA neurons is illustrated in e.
    Alexa Fluor 594 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2663 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 568 goat anti rabbit igg
    Conjugate treatment reduced caspase-3 protein expression in HSG cells. HSG cells were treated with 8.71μM conjugate and incubated for 72 hours. Caspase-3 protein expression was analyzed by immunofluorescence (A) using rabbit anti-caspase-3 antibodies and <t>alexa</t> <t>fluor</t> 568 goat anti-rabbit <t>IgG</t> secondary antibodies (red). Cell nuclei were counterstained with DAPI (blue), and caspase-3 siRNA or conjugate was detected by in situ hybridization (green). Images shown at 100X magnification. (B) Quantitation of caspase-3 protein levels were measured using Image J image analysis software and normalized to untreated cells. Asterisks (*) indicate p
    Alexa Fluor 568 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1541 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated goat anti rabbit igg
    Expression of HEV cysteine protease. (A) It represents PCR amplification of Cysteine Protease from BacMam-HEV infected cells using primers G3CPF and G3CPR ( Supplementary Table S1 ). The corresponding lanes are as follows: Marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blotting analysis using Anti-Cysteine Protease antibody and Goat Anti-Rabbit <t>IgG</t> <t>HRP</t> conjugated secondary antibody. The corresponding lanes are as follows: Marker (lane 1), cell lysate from Huh7 control cells (lane 2), cell lysate from infected Huh7 cells showing specific band identical to the size of HEV Cysteine Protease (lane 3). (C) Immunofluorescence assay: Cells were stained with Anti-Cysteine Protease primary antibody, which was detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh7 cells and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. The statistical significance was tested by the Mann–Whitney test ( p -value = 0.0286). * indicates p -value
    Hrp Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 conjugated goat anti rabbit igg
    Subcellular colocalization of endogenous ZNF265 with endogenous nuclear factors. (A) Immunoblotting assay demonstrates specific recognition of ZNF265 by the polyclonal ZNF265 antibody (the arrow shows a 55-kD band), which was competed by ZNF265 oligopeptide antigen (2.5 μg/ml) in three replicate experiments. (B) Subcellular localization of various protein factors. Fixed Calu-6 cells were exposed to: (1st column) monoclonal antibodies against splicing factors U1-70K, Sm antigen, SC35, SMN, or transcriptosomal factors p300 and YY1, in respective rows, before incubation with <t>Alexa</t> 594 anti–mouse <t>IgG</t> (red); (2nd column) staining with anti-ZNF265 and detection with Alexa 488–conjugated anti–rabbit IgG (green); (3rd column) DAPI staining of nucleus (blue); (4th column) digital overlay of Z-series projections shown in columns 1 and 2 to demonstrate colocalization (yellow); (5th column) scattergrams of the overlayed projection shown in column 4. Each row represents the same field (width × height = 60 × 60 μm), acquired using three-channel confocal microscopy.
    Alexa Fluor 594 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa 488 conjugated goat anti rabbit igg
    Binding of HSV-1 gD:Fc and PRV-gD:Fc to MDCK cells (A, C, E, and G) and MDCK-nectin-1 cells (B, D, F, and H). The cells were maintained in NC medium (A to D) or switched to LC medium for 2 h (E to H) and then incubated with either HSV-1 gD:Fc (A, B, E, F, I, and J) or PRV gD:Fc (C, D, G, and H), followed by fixation and incubation with Alexa 488-conjugated goat anti-rabbit <t>IgG.</t> (I and J) Orthogonal sections of HSV-1 gD:Fc-stained MDCK-nectin-1 cells maintained in NC medium (I) or LC medium for 2 h (J). Bar, 10 μm.
    Alexa 488 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher peroxidase conjugated goat anti rabbit igg
    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to <t>NOD2-mediated</t> signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control <t>IgG,</t> solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P
    Peroxidase Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to <t>NOD2-mediated</t> signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control <t>IgG,</t> solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg h l superclonal secondary antibody
    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to <t>NOD2-mediated</t> signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control <t>IgG,</t> solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P
    Goat Anti Rabbit Igg H L Superclonal Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg hrp
    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to <t>NOD2-mediated</t> signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control <t>IgG,</t> solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P
    Goat Anti Rabbit Igg Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 555 goat anti rabbit igg
    Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. <t>Alexa</t> Fluor 488 (green) and Alexa <t>Fluor</t> 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.
    Alexa Fluor 555 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 568 conjugated goat anti rabbit igg
    Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human <t>IgG</t> labeled with <t>Alexa</t> Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.
    Alexa Fluor 568 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1012 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti rabbit igg
    Apoptosis-modulating ϕ mutations alter μ1 distribution in cells. (A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse <t>IgG</t> conjugated to <t>Alexa</t> Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P
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    Thermo Fisher alexa fluor 647 goat anti rabbit igg
    Immunofluorescence on cells expressing wild-type or chimeric rmGlu 6 or rmGlu 7 constructs. Cells were stained after fixation and permeabilization with <t>MAB1/28-IgG</t> followed by secondary antibody labelled with <t>Alexa</t> Fluor 647. In the top panel the predicted receptor topology is indicated relative to the membrane with the large extracellular domain at the top, with the mGlu 6 part in grey and mGlu 7 in black. Shown are representative overlaid images of immunostain (green) and nuclear stain (blue) acquired at 20 × magnification using same exposure parameters for the different are shown.
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    Image Search Results


    Loss of VCP induces EMT A. 72 hours after transfection of A549, H358 or immortalized human bronchiolar HPLD-1 cells with siRNA against VCP or non-targeting cell lysates were prepared and western blot analysis of EMT markers Vimentin and E-cadherin was performed. B. Fluorescence staining for E-cadherin and Vimentin in A549 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. C. A549 cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Journal: Oncotarget

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress

    doi:

    Figure Lengend Snippet: Loss of VCP induces EMT A. 72 hours after transfection of A549, H358 or immortalized human bronchiolar HPLD-1 cells with siRNA against VCP or non-targeting cell lysates were prepared and western blot analysis of EMT markers Vimentin and E-cadherin was performed. B. Fluorescence staining for E-cadherin and Vimentin in A549 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides and stained for EMT markers. i, iii and v: E-cadherin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). ii, iv and vi: overlay of respective E-cadherin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Vimentin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Vimentin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. C. A549 cells were prepared as described in B and F-actin was detected with Alexa Fluor 568 Phalloidin (red). Re-organization of actin cytoskeleton through destruction and cellular protrusion formation is indicated by arrows.

    Article Snippet: USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Techniques: Transfection, Western Blot, Fluorescence, Staining

    Loss of VCP induces ER stress A. Western blot analysis of different proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. B. Fluorescence staining for Chop and Calnexin in A549 and H358 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides. After 48 hours cells were fixed and stained for Chop and Calnexin. i, iii and v: Chop was detected using Alexa Fluor 488 rabbit anti-mouse IgG (green). ii, iv and vi: overlay of respective Chop and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Calnexin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Calnexin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain.

    Journal: Oncotarget

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress

    doi:

    Figure Lengend Snippet: Loss of VCP induces ER stress A. Western blot analysis of different proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. B. Fluorescence staining for Chop and Calnexin in A549 and H358 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides. After 48 hours cells were fixed and stained for Chop and Calnexin. i, iii and v: Chop was detected using Alexa Fluor 488 rabbit anti-mouse IgG (green). ii, iv and vi: overlay of respective Chop and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Calnexin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Calnexin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain.

    Article Snippet: USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Techniques: Western Blot, Transfection, Expressing, Fluorescence, Staining

    Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p

    Article Snippet: Cells were then washed with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific) at 1:200 dilution for 1 h at room temperature.

    Techniques: Labeling, Recombinant, Incubation

    Immunostaining of wing imaginal discs with anti-Lsd1 antibody. ( A , B ) wing imaginal discs were reacted with rabbit anti-Lsd1 antibody followed by anti-rabbit IgG Alexa Fluor™ 488 antibody; MS1096-GAL4 flies ( A ) showed signal clearly and MS1096-GAL4 > UAS- Lsd1 IR flies ( B ) showed decreased Lsd1 signal in the wing pouch of wing imaginal discs ( C , D ) immunostaining of wing imaginal discs with only the anti-rabbit IgG Alexa Fluor TM 488 antibody showed no detectable signal; ( C ) MS1096-GAL4; ( D ) MS1096-GAL4 > UAS- Lsd1 IR. Flies were reared at 25 °C. The circles indicate wing pouch of wing discs where Lsd1 was knocked down.

    Journal: International Journal of Molecular Sciences

    Article Title: Function of Lipid Storage Droplet 1 (Lsd1) in Wing Development of Drosophila melanogaster

    doi: 10.3390/ijms17050648

    Figure Lengend Snippet: Immunostaining of wing imaginal discs with anti-Lsd1 antibody. ( A , B ) wing imaginal discs were reacted with rabbit anti-Lsd1 antibody followed by anti-rabbit IgG Alexa Fluor™ 488 antibody; MS1096-GAL4 flies ( A ) showed signal clearly and MS1096-GAL4 > UAS- Lsd1 IR flies ( B ) showed decreased Lsd1 signal in the wing pouch of wing imaginal discs ( C , D ) immunostaining of wing imaginal discs with only the anti-rabbit IgG Alexa Fluor TM 488 antibody showed no detectable signal; ( C ) MS1096-GAL4; ( D ) MS1096-GAL4 > UAS- Lsd1 IR. Flies were reared at 25 °C. The circles indicate wing pouch of wing discs where Lsd1 was knocked down.

    Article Snippet: After extensive washing with PBST, the samples were incubated with goat anti-rabbit IgG Alexa Fluor™ 488 (Molecular Probes, Invitrogen) at a 1:400 dilution for 2 h at 25 °C, washed with PBST and PBS, and then mounted in Vectashield mounting medium.

    Techniques: Immunostaining

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.

    Journal: The Journal of Infectious Diseases

    Article Title: Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA

    doi: 10.1093/infdis/jiy467

    Figure Lengend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.

    Article Snippet: Rabbit anti-VAR2CSA or normal rabbit serum was preabsorbed on uninfected RBCs, incubated with infected RBCs at a ratio of 1:40, and detected with AlexaFluor 647–conjugated goat anti-rabbit IgG (dilution, 1:500; Life Technologies).

    Techniques: Binding Assay, Infection, Inhibition, Incubation

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.

    Journal: The Journal of Infectious Diseases

    Article Title: Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA

    doi: 10.1093/infdis/jiy467

    Figure Lengend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.

    Article Snippet: Rabbit anti-VAR2CSA or normal rabbit serum was preabsorbed on uninfected RBCs, incubated with infected RBCs at a ratio of 1:40, and detected with AlexaFluor 647–conjugated goat anti-rabbit IgG (dilution, 1:500; Life Technologies).

    Techniques: Binding Assay, Infection, Flow Cytometry, Cytometry, Expressing, Staining

    Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with Alexa Fluor 488-conjugated anti-rabbit

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

    doi: 10.1073/pnas.1208703109

    Figure Lengend Snippet: Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with Alexa Fluor 488-conjugated anti-rabbit

    Article Snippet: Briefly, dissected guts from wandering larvae or adult females were fixed in 4% (wt/vol) paraformaldehyde in PBS solution for 1 h, sections were incubated for 2 h with partially purified MCO1 antiserum or preimmune serum, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) was used as the secondary antibody.

    Techniques:

    Drosophila lacking dLRRK kinase activity has normal development of DA neuron . Brain dissected from wild-type flies (a), e03680/+ flies (b) and e03680/e03680 flies (c) aged 20 days were immunostained with anti- Drosophila TH antibody followed by an Alexa Fluor 594-labeled secondary antibody to indentify DA neurons. Representative pictures shown were collected by confocal microscopy. Dopaminergic neurons in six brain regions, including PAL, PPM1/2, PPM3, PPL1, PPL2, and VUM, were quantified and no significant difference was found among different fly lines (d). Localization of Drosophila DA neurons is illustrated in e.

    Journal: Molecular Neurodegeneration

    Article Title: Dispensable role of Drosophila ortholog of LRRK2 kinase activity in survival of dopaminergic neurons

    doi: 10.1186/1750-1326-3-3

    Figure Lengend Snippet: Drosophila lacking dLRRK kinase activity has normal development of DA neuron . Brain dissected from wild-type flies (a), e03680/+ flies (b) and e03680/e03680 flies (c) aged 20 days were immunostained with anti- Drosophila TH antibody followed by an Alexa Fluor 594-labeled secondary antibody to indentify DA neurons. Representative pictures shown were collected by confocal microscopy. Dopaminergic neurons in six brain regions, including PAL, PPM1/2, PPM3, PPL1, PPL2, and VUM, were quantified and no significant difference was found among different fly lines (d). Localization of Drosophila DA neurons is illustrated in e.

    Article Snippet: Anti-Drosophila TH antibody (1:500) was generously provided by Dr. Neckameyer (Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104), Alexa Fluor 594 goat anti-rabbit IgG was from Invitrogen (San Diego, CA).

    Techniques: Activity Assay, Labeling, Confocal Microscopy

    Conjugate treatment reduced caspase-3 protein expression in HSG cells. HSG cells were treated with 8.71μM conjugate and incubated for 72 hours. Caspase-3 protein expression was analyzed by immunofluorescence (A) using rabbit anti-caspase-3 antibodies and alexa fluor 568 goat anti-rabbit IgG secondary antibodies (red). Cell nuclei were counterstained with DAPI (blue), and caspase-3 siRNA or conjugate was detected by in situ hybridization (green). Images shown at 100X magnification. (B) Quantitation of caspase-3 protein levels were measured using Image J image analysis software and normalized to untreated cells. Asterisks (*) indicate p

    Journal: Arthritis and Rheumatism

    Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

    doi: 10.1002/art.30450

    Figure Lengend Snippet: Conjugate treatment reduced caspase-3 protein expression in HSG cells. HSG cells were treated with 8.71μM conjugate and incubated for 72 hours. Caspase-3 protein expression was analyzed by immunofluorescence (A) using rabbit anti-caspase-3 antibodies and alexa fluor 568 goat anti-rabbit IgG secondary antibodies (red). Cell nuclei were counterstained with DAPI (blue), and caspase-3 siRNA or conjugate was detected by in situ hybridization (green). Images shown at 100X magnification. (B) Quantitation of caspase-3 protein levels were measured using Image J image analysis software and normalized to untreated cells. Asterisks (*) indicate p

    Article Snippet: Secondary antibodies used were Alexa Fluor 568 goat anti-rabbit IgG (1:400) from Molecular Probes (Carlsbad, CA).

    Techniques: Expressing, Incubation, Immunofluorescence, In Situ Hybridization, Quantitation Assay, Software

    Expression of HEV cysteine protease. (A) It represents PCR amplification of Cysteine Protease from BacMam-HEV infected cells using primers G3CPF and G3CPR ( Supplementary Table S1 ). The corresponding lanes are as follows: Marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blotting analysis using Anti-Cysteine Protease antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: Marker (lane 1), cell lysate from Huh7 control cells (lane 2), cell lysate from infected Huh7 cells showing specific band identical to the size of HEV Cysteine Protease (lane 3). (C) Immunofluorescence assay: Cells were stained with Anti-Cysteine Protease primary antibody, which was detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh7 cells and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. The statistical significance was tested by the Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Journal: Frontiers in Microbiology

    Article Title: Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing

    doi: 10.3389/fmicb.2020.01347

    Figure Lengend Snippet: Expression of HEV cysteine protease. (A) It represents PCR amplification of Cysteine Protease from BacMam-HEV infected cells using primers G3CPF and G3CPR ( Supplementary Table S1 ). The corresponding lanes are as follows: Marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blotting analysis using Anti-Cysteine Protease antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: Marker (lane 1), cell lysate from Huh7 control cells (lane 2), cell lysate from infected Huh7 cells showing specific band identical to the size of HEV Cysteine Protease (lane 3). (C) Immunofluorescence assay: Cells were stained with Anti-Cysteine Protease primary antibody, which was detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh7 cells and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. The statistical significance was tested by the Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Article Snippet: Subsequently, the membrane was washed with 5 ml of PBST and incubated with 1:3000 dilutions of HRP-conjugated goat Anti-Rabbit IgG (Invitrogen) and developed with DAB (Sigma) or with Clarity, Max ECL Western blotting substrate (Bio-Rad).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Infection, Marker, Western Blot, Immunofluorescence, Staining, Fluorescence, Standard Deviation, MANN-WHITNEY

    Expression of active methyltransferase. (A) PCR Amplification of methyltransferase from BacMam-HEV infected cells using primers G3MeTF and G3MeTR ( Supplementary Table S1 ). The corresponding lanes are as follows: Marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blot analysis using anti-methyltransferase antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: Marker (lane 1), cell lysate from Huh 7 control cells (lane 2), cell lysate from infected Huh 7 cells showing specific band identical to the size of methyltransferase (lane 3). (C) Immunofluorescence assay: cells were stained with Anti-MeT detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh7 cells, and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image. The Immunofluorescence was performed three times as independent experiments. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. Statistical significance of data was determined by the Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Journal: Frontiers in Microbiology

    Article Title: Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing

    doi: 10.3389/fmicb.2020.01347

    Figure Lengend Snippet: Expression of active methyltransferase. (A) PCR Amplification of methyltransferase from BacMam-HEV infected cells using primers G3MeTF and G3MeTR ( Supplementary Table S1 ). The corresponding lanes are as follows: Marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blot analysis using anti-methyltransferase antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: Marker (lane 1), cell lysate from Huh 7 control cells (lane 2), cell lysate from infected Huh 7 cells showing specific band identical to the size of methyltransferase (lane 3). (C) Immunofluorescence assay: cells were stained with Anti-MeT detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh7 cells, and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image. The Immunofluorescence was performed three times as independent experiments. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. Statistical significance of data was determined by the Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Article Snippet: Subsequently, the membrane was washed with 5 ml of PBST and incubated with 1:3000 dilutions of HRP-conjugated goat Anti-Rabbit IgG (Invitrogen) and developed with DAB (Sigma) or with Clarity, Max ECL Western blotting substrate (Bio-Rad).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Infection, Marker, Western Blot, Immunofluorescence, Staining, Fluorescence, Standard Deviation, MANN-WHITNEY

    Expression of RNA-dependent RNA polymerase. (A) PCR amplification of RdRp from BacMam-HEV infected cells using RdRp specific primers ( Supplementary Table S1 ). The corresponding lanes are as follows: marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blot analysis using anti-RdRp Antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: marker (lane 1), cell lysate from Huh7 control cells (lane 2), cell lysate from infected Huh7 cells showing specific band identical to the size of HEV RNA dependent RNA polymerase (lane 3). (C) Immunofluorescence assay: cells were stained with Anti-RdRp Primary Antibody detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh cells and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image of (a,b) and (d,f) , respectively. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. The data was significantly tested using Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Journal: Frontiers in Microbiology

    Article Title: Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing

    doi: 10.3389/fmicb.2020.01347

    Figure Lengend Snippet: Expression of RNA-dependent RNA polymerase. (A) PCR amplification of RdRp from BacMam-HEV infected cells using RdRp specific primers ( Supplementary Table S1 ). The corresponding lanes are as follows: marker (lane 1), cDNA from uninfected cells (lane 2), cDNA from infected cells (lane 3). (B) Western blot analysis using anti-RdRp Antibody and Goat Anti-Rabbit IgG HRP conjugated secondary antibody. The corresponding lanes are as follows: marker (lane 1), cell lysate from Huh7 control cells (lane 2), cell lysate from infected Huh7 cells showing specific band identical to the size of HEV RNA dependent RNA polymerase (lane 3). (C) Immunofluorescence assay: cells were stained with Anti-RdRp Primary Antibody detected with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody and counterstained with DAPI to stain nuclei. Panels (a–c) represent control Huh cells and panels (d–f) represent HEV infected cells. Panels (c,f) represent the merged image of (a,b) and (d,f) , respectively. (D) The graph represents the mean fluorescence intensity, and the error bar indicates the standard deviation. The data was significantly tested using Mann–Whitney test ( p -value = 0.0286). * indicates p -value

    Article Snippet: Subsequently, the membrane was washed with 5 ml of PBST and incubated with 1:3000 dilutions of HRP-conjugated goat Anti-Rabbit IgG (Invitrogen) and developed with DAB (Sigma) or with Clarity, Max ECL Western blotting substrate (Bio-Rad).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Infection, Marker, Western Blot, Immunofluorescence, Staining, Fluorescence, Standard Deviation, MANN-WHITNEY

    Subcellular colocalization of endogenous ZNF265 with endogenous nuclear factors. (A) Immunoblotting assay demonstrates specific recognition of ZNF265 by the polyclonal ZNF265 antibody (the arrow shows a 55-kD band), which was competed by ZNF265 oligopeptide antigen (2.5 μg/ml) in three replicate experiments. (B) Subcellular localization of various protein factors. Fixed Calu-6 cells were exposed to: (1st column) monoclonal antibodies against splicing factors U1-70K, Sm antigen, SC35, SMN, or transcriptosomal factors p300 and YY1, in respective rows, before incubation with Alexa 594 anti–mouse IgG (red); (2nd column) staining with anti-ZNF265 and detection with Alexa 488–conjugated anti–rabbit IgG (green); (3rd column) DAPI staining of nucleus (blue); (4th column) digital overlay of Z-series projections shown in columns 1 and 2 to demonstrate colocalization (yellow); (5th column) scattergrams of the overlayed projection shown in column 4. Each row represents the same field (width × height = 60 × 60 μm), acquired using three-channel confocal microscopy.

    Journal: The Journal of Cell Biology

    Article Title: ZNF265--a novel spliceosomal protein able to induce alternative splicing

    doi: 10.1083/jcb.200010059

    Figure Lengend Snippet: Subcellular colocalization of endogenous ZNF265 with endogenous nuclear factors. (A) Immunoblotting assay demonstrates specific recognition of ZNF265 by the polyclonal ZNF265 antibody (the arrow shows a 55-kD band), which was competed by ZNF265 oligopeptide antigen (2.5 μg/ml) in three replicate experiments. (B) Subcellular localization of various protein factors. Fixed Calu-6 cells were exposed to: (1st column) monoclonal antibodies against splicing factors U1-70K, Sm antigen, SC35, SMN, or transcriptosomal factors p300 and YY1, in respective rows, before incubation with Alexa 594 anti–mouse IgG (red); (2nd column) staining with anti-ZNF265 and detection with Alexa 488–conjugated anti–rabbit IgG (green); (3rd column) DAPI staining of nucleus (blue); (4th column) digital overlay of Z-series projections shown in columns 1 and 2 to demonstrate colocalization (yellow); (5th column) scattergrams of the overlayed projection shown in column 4. Each row represents the same field (width × height = 60 × 60 μm), acquired using three-channel confocal microscopy.

    Article Snippet: Secondary antibodies used were: Alexa Fluor 488–conjugated goat anti–mouse IgG (Molecular Probes), Alexa Fluor 594–conjugated goat anti–rabbit IgG (Molecular Probes), alkaline phosphatase–conjugated rabbit anti–mouse IgG (Sigma-Aldrich), and alkaline phosphatase–conjugated goat anti–rabbit IgG (Sigma-Aldrich).

    Techniques: Incubation, Staining, Confocal Microscopy

    Binding of HSV-1 gD:Fc and PRV-gD:Fc to MDCK cells (A, C, E, and G) and MDCK-nectin-1 cells (B, D, F, and H). The cells were maintained in NC medium (A to D) or switched to LC medium for 2 h (E to H) and then incubated with either HSV-1 gD:Fc (A, B, E, F, I, and J) or PRV gD:Fc (C, D, G, and H), followed by fixation and incubation with Alexa 488-conjugated goat anti-rabbit IgG. (I and J) Orthogonal sections of HSV-1 gD:Fc-stained MDCK-nectin-1 cells maintained in NC medium (I) or LC medium for 2 h (J). Bar, 10 μm.

    Journal: Journal of Virology

    Article Title: Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

    doi: 10.1128/JVI.76.14.7203-7208.2002

    Figure Lengend Snippet: Binding of HSV-1 gD:Fc and PRV-gD:Fc to MDCK cells (A, C, E, and G) and MDCK-nectin-1 cells (B, D, F, and H). The cells were maintained in NC medium (A to D) or switched to LC medium for 2 h (E to H) and then incubated with either HSV-1 gD:Fc (A, B, E, F, I, and J) or PRV gD:Fc (C, D, G, and H), followed by fixation and incubation with Alexa 488-conjugated goat anti-rabbit IgG. (I and J) Orthogonal sections of HSV-1 gD:Fc-stained MDCK-nectin-1 cells maintained in NC medium (I) or LC medium for 2 h (J). Bar, 10 μm.

    Article Snippet: Alternatively, cells grown on coverslips were incubated with gD:Fc, and binding was visualized with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes).

    Techniques: Binding Assay, Incubation, Staining

    SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P

    Journal: PLoS Pathogens

    Article Title: NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis

    doi: 10.1371/journal.ppat.1003351

    Figure Lengend Snippet: SB203580, an RIP2 inhibitor downstream of nucleotide-binding oligomerization domain (NOD)2, attenuates CLP-induced sepsis. (A) Peritoneal cells of WT mice were cultured with SB203580 and/or MDP for 24 h, and IL-1β and IL-10 concentrations were measured in culture fractions. (B) Molecules related to NOD2-mediated signal transduction were blotted using peritoneal cells obtained from WT and Nod2 −/− mice injected with SB203580 or PBS 24 h after CLP. (C) Serum and peritoneal IL-1β, IL-10, and C5a levels were estimated in WT (n = 4) and Nod2 −/− (n = 3) mice injected with SB203580 (n = 4 in WT, n = 3 in Nod2 −/− ) or PBS 24 h after CLP by ELISA. (D) The levels of CD55 expression on F4/80 − Ly6-G + cells from WT (n = 3) and WT mice injected with SB203580 (n = 3) were measured 24 h after CLP. (mean fluorescence intensity [MFI] of CD55 expression in the panels, diagrams filled with gray for istotype-matched control IgG, solid lines for CD55) (E) The percentages of surviving mice were estimated during CLP-induced sepsis ( a P = 0.0312, log-rank test, n = 6–8 per group; WT mice injected with SB203580 vs. PBS). *P

    Article Snippet: Antibodies against phosphor–p38, p38 (Cell Signaling Technology, MA, USA), phosphor-Rip2 (Thermo scientific, Rockford, USA), Rip2 (Santa Cruz Biotechnology, CA, USA), Bb (Santa Cruz Biotechnology), NOD2 (Santa Cruz Biotechnology), and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Thermo scientific) were used.

    Techniques: Binding Assay, Mouse Assay, Cell Culture, Transduction, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

    IL-1β-dependent IL-10 production mediated by nucleotide-binding oligomerization domain (NOD) 2 enhances C5a generation by suppressing CD55 expression on Ly6-G + cells during sepsis. (A) CD55 and CR1/2 expression on gated F4/80 − Ly6-G + peritoneal cells from WT, Nod2 −/− , and Nod2 −/− mice injected with recombinant IL-1β or IL-10 was estimated 24 h after CLP (mean fluorescence intensity [MFI] of CD55 expression in the panels). (B) CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from Il-10 −/− or Il-10 −/− mice injected with recombinant IL-1β was estimated 24 h after CLP (MFI of CD55 expression in the panels) (C) To block IL-10 receptor engagement in vivo , anti-IL10 receptor mAbs were i.p. injected into WT and Nod2 −/− mice administered recombinant IL-10 during CLP-induced sepsis. CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from these mice 24 h after CLP was evaluated. (D) The levels of CD55 expression on F4/80 − Ly6-G + peritoneal cells were compared in WT, Nod2 −/− , IL-10 −/− , and Il-1r −/− mice 24 h after CLP. (A–D) anti-CD55 mAb (lines) and control IgG (diagrams filled with gray) were used. (E) Peritoneal cells from WT and Nod2 −/− mice 24 h after CLP were blotted for Bb factor. (F) Peritoneal cells from WT and Nod2 −/− mice 12 h after CLP were incubated with RPMI media containing 10% WT mouse serum for 24 h. (G and H) To evaluate the effect of CD55 on C5a generation in vivo , WT, Nod2 −/− , (G) or Nod2 −/− mice given recombinant IL-10 (H) were i.p. injected with soluble CD55 12 h after CLP. Serum and peritoneal C5a levels and the survival percentages of these mice were measured during CLP-induced sepsis ( a P = 0.0124, log-rank test; WT [n = 8], Nod2 −/− [n = 8 ], and soluble CD55-injected WT [n = 6 ] or Nod2 −/− mice [n = 8 ] in G, a P = 0.0024, log-rank test; Nod2 −/− mice [n = 8], Nod2 −/− mice injected with recombinant IL-10 [n = 8] or recombinant IL-10 and soluble CD55 [n = 6] in H). *P

    Journal: PLoS Pathogens

    Article Title: NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis

    doi: 10.1371/journal.ppat.1003351

    Figure Lengend Snippet: IL-1β-dependent IL-10 production mediated by nucleotide-binding oligomerization domain (NOD) 2 enhances C5a generation by suppressing CD55 expression on Ly6-G + cells during sepsis. (A) CD55 and CR1/2 expression on gated F4/80 − Ly6-G + peritoneal cells from WT, Nod2 −/− , and Nod2 −/− mice injected with recombinant IL-1β or IL-10 was estimated 24 h after CLP (mean fluorescence intensity [MFI] of CD55 expression in the panels). (B) CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from Il-10 −/− or Il-10 −/− mice injected with recombinant IL-1β was estimated 24 h after CLP (MFI of CD55 expression in the panels) (C) To block IL-10 receptor engagement in vivo , anti-IL10 receptor mAbs were i.p. injected into WT and Nod2 −/− mice administered recombinant IL-10 during CLP-induced sepsis. CD55 expression on gated F4/80 − Ly6-G + peritoneal cells from these mice 24 h after CLP was evaluated. (D) The levels of CD55 expression on F4/80 − Ly6-G + peritoneal cells were compared in WT, Nod2 −/− , IL-10 −/− , and Il-1r −/− mice 24 h after CLP. (A–D) anti-CD55 mAb (lines) and control IgG (diagrams filled with gray) were used. (E) Peritoneal cells from WT and Nod2 −/− mice 24 h after CLP were blotted for Bb factor. (F) Peritoneal cells from WT and Nod2 −/− mice 12 h after CLP were incubated with RPMI media containing 10% WT mouse serum for 24 h. (G and H) To evaluate the effect of CD55 on C5a generation in vivo , WT, Nod2 −/− , (G) or Nod2 −/− mice given recombinant IL-10 (H) were i.p. injected with soluble CD55 12 h after CLP. Serum and peritoneal C5a levels and the survival percentages of these mice were measured during CLP-induced sepsis ( a P = 0.0124, log-rank test; WT [n = 8], Nod2 −/− [n = 8 ], and soluble CD55-injected WT [n = 6 ] or Nod2 −/− mice [n = 8 ] in G, a P = 0.0024, log-rank test; Nod2 −/− mice [n = 8], Nod2 −/− mice injected with recombinant IL-10 [n = 8] or recombinant IL-10 and soluble CD55 [n = 6] in H). *P

    Article Snippet: Antibodies against phosphor–p38, p38 (Cell Signaling Technology, MA, USA), phosphor-Rip2 (Thermo scientific, Rockford, USA), Rip2 (Santa Cruz Biotechnology, CA, USA), Bb (Santa Cruz Biotechnology), NOD2 (Santa Cruz Biotechnology), and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Thermo scientific) were used.

    Techniques: Binding Assay, Expressing, Mouse Assay, Injection, Recombinant, Fluorescence, Blocking Assay, In Vivo, Incubation

    Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.

    Journal: Journal of Molecular Cell Biology

    Article Title: Tubulin detyrosination promotes human trophoblast syncytium formation

    doi: 10.1093/jmcb/mjz084

    Figure Lengend Snippet: Membrane proteins required for fusion are enriched in detyr-α-tub-overexpressing cell membranes. ( A ) Time-lapse images of BeWo cells carrying EGFP-α-tubulin or mCherry-α-tubulin. The cells were treated with forskolin to induce fusion. The white arrowheads indicate where fusion occurred. Scale bar, 10 μm. ( B ) SR-SIM. Alexa Fluor 488 (green) and Alexa Fluor 555 (red) were used to label α-tubulin and detyr-α-tub, respectively, in cells after a 48-h forskolin treatment. Scale bar, 2.5 μm. ( C ) The influences of TTL overexpression or knockdown on the expression of cell surface proteins in different cell fractions of BeWo cells treated with forskolin. The cell surface proteins were isolated and examined with the indicated antibodies. Na + /K + -ATPase served as a plasma membrane loading control. Whole-cell lysates were harvested and examined with the indicated antibodies. GAPDH served as a loading control.

    Article Snippet: After incubation at 4°C overnight with the primary antibodies, the cells were washed and incubated with the appropriate highly cross-adsorbed Alexa Fluor 488 goat anti-mouse IgG (A-11001, RRID: AB_2534069, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (A-21422, RRID: AB_2535844, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (A-11034, RRID: AB_2576217, Thermo Fisher Scientific), or Alexa Fluor 555 goat anti-rabbit IgG (A-21428, RRID: AB_2535849, Thermo Fisher Scientific).

    Techniques: Over Expression, Expressing, Isolation

    Detyrosination of α-tubulin varies during syncytialization of primary CTBs from normal and PE placentae. ( A ) Western blotting of primary CTBs isolated from both normal and PE placentae after spontaneous syncytialization for 72 h using anti-detyr-α-tub, anti-TTL, anti-α-tubulin, anti-Syncytin-2, and anti-GAPDH antibodies. The colored lines represent groups of corresponding gestational ages. ( B ) Quantification of detyr-α-tub from the western blotting data in A . The band intensities detected with anti-detyr-α-tub antibodies were normalized to the band intensity of GAPDH as the loading control. ( C ) The fusion index was determined contemporaneously with the samples harvested for western blotting in A . For confocal microscopy, Alexa Fluor 555 (red) was used to label E-cadherin to identify the cell boundaries. Nuclei were stained with DAPI (blue). The dots and squares representing individual samples from patients are colored according to the matched gestational ages. The horizontal lines represent the means. ( D ) Primary CTBs from preeclamptic placentae undergoing in vitro syncytialization were simultaneously treated with TTL siRNA and subjected to western blotting analysis using anti-detyr-α-tub, anti-TTL, and anti-GAPDH antibodies. The fusion indices were determined as in C . The lines in the enrichment and fusion index results represent groups of PE placentae and normal placentae of corresponding gestational ages. Scale bar, 20 μm.

    Journal: Journal of Molecular Cell Biology

    Article Title: Tubulin detyrosination promotes human trophoblast syncytium formation

    doi: 10.1093/jmcb/mjz084

    Figure Lengend Snippet: Detyrosination of α-tubulin varies during syncytialization of primary CTBs from normal and PE placentae. ( A ) Western blotting of primary CTBs isolated from both normal and PE placentae after spontaneous syncytialization for 72 h using anti-detyr-α-tub, anti-TTL, anti-α-tubulin, anti-Syncytin-2, and anti-GAPDH antibodies. The colored lines represent groups of corresponding gestational ages. ( B ) Quantification of detyr-α-tub from the western blotting data in A . The band intensities detected with anti-detyr-α-tub antibodies were normalized to the band intensity of GAPDH as the loading control. ( C ) The fusion index was determined contemporaneously with the samples harvested for western blotting in A . For confocal microscopy, Alexa Fluor 555 (red) was used to label E-cadherin to identify the cell boundaries. Nuclei were stained with DAPI (blue). The dots and squares representing individual samples from patients are colored according to the matched gestational ages. The horizontal lines represent the means. ( D ) Primary CTBs from preeclamptic placentae undergoing in vitro syncytialization were simultaneously treated with TTL siRNA and subjected to western blotting analysis using anti-detyr-α-tub, anti-TTL, and anti-GAPDH antibodies. The fusion indices were determined as in C . The lines in the enrichment and fusion index results represent groups of PE placentae and normal placentae of corresponding gestational ages. Scale bar, 20 μm.

    Article Snippet: After incubation at 4°C overnight with the primary antibodies, the cells were washed and incubated with the appropriate highly cross-adsorbed Alexa Fluor 488 goat anti-mouse IgG (A-11001, RRID: AB_2534069, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (A-21422, RRID: AB_2535844, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (A-11034, RRID: AB_2576217, Thermo Fisher Scientific), or Alexa Fluor 555 goat anti-rabbit IgG (A-21428, RRID: AB_2535849, Thermo Fisher Scientific).

    Techniques: Western Blot, Isolation, Confocal Microscopy, Staining, In Vitro

    Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.

    Journal: Journal of Molecular Cell Biology

    Article Title: Tubulin detyrosination promotes human trophoblast syncytium formation

    doi: 10.1093/jmcb/mjz084

    Figure Lengend Snippet: Augmented detyrosination of α-tubulin during spontaneous fusion of primary CTBs. ( A ) Illustrative model depicting BeWo cell fusion. ( B ) Upper panel: schematic illustration of spontaneous syncytialization of human primary CTBs after isolation. Lower panel: human trophoblasts stained at 0 and 72 h of culture for E-cadherin and nuclei. Scale bar, 50 μm. ( C ) Results of western blotting using the indicated antibodies on primary CTBs from the indicated gestational ages and their corresponding syncytia after 72 h of incubation. GAPDH was used as a loading control (here and hereafter). The colored lines represent groups of corresponding gestational ages. ( D ) Quantification of the western blot data in C . The band intensities detected with anti-α-tubulin and anti-detyr-α-tub antibodies were normalized to that of GAPDH as a control. The dots and squares representing individual samples from patients are colored according to the matching samples at different time points. The horizontal lines represent the means. ( E ) For confocal microscopy, Alexa Fluor 488 (green) was used to label TTL. Alexa Fluor 555 (red) was used to label Syncytin-2, active caspase-8, or CK7. Scale bar, 10 μm. The arrows indicate a layer of mononucleated CTBs and a layer of multinucleated STB.

    Article Snippet: After incubation at 4°C overnight with the primary antibodies, the cells were washed and incubated with the appropriate highly cross-adsorbed Alexa Fluor 488 goat anti-mouse IgG (A-11001, RRID: AB_2534069, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (A-21422, RRID: AB_2535844, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (A-11034, RRID: AB_2576217, Thermo Fisher Scientific), or Alexa Fluor 555 goat anti-rabbit IgG (A-21428, RRID: AB_2535849, Thermo Fisher Scientific).

    Techniques: Isolation, Staining, Western Blot, Incubation, Confocal Microscopy

    Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.

    Journal: Journal of Virology

    Article Title: Hepatitis E Virus Genotype 1 Infection of Swine Kidney Cells In Vitro Is Inhibited at Multiple Levels

    doi: 10.1128/JVI.02205-13

    Figure Lengend Snippet: Flow cytometry of transfected cells stained separately for the ORF2 or ORF3 protein. HepG2/C3A and LLC-PK cells electroporated with transcripts from P6 (A), Sar55 (B), or Sar/S17 (C, D) were plated in 6 wells of a 6-well culture plate, incubated at 34.5°C, and harvested 7 (A, B) or 5 (C, D) days later. Cells in triplicate wells were stained for the ORF2 protein, followed by goat anti-human IgG labeled with Alexa Fluor 488 (open bars); cells in the other 3 wells were stained for the ORF3 protein, followed by goat anti-rabbit IgG also labeled with Alexa Fluor 488 (hatched bars). Flow cytometry was performed with the same settings for all samples. (A to C) Mean percentage of positive cells, in triplicate; (D) mean of the geometric mean fluorescence intensity in the same triplicate samples assayed for panel C. Shaded bars, ORF2; dotted bars, ORF3. Error bars indicate standard deviations. P values were determined by Student's t test; P values of less than 0.05 were statistically significant.

    Article Snippet: Secondary antibodies were a mixture of Alexa Fluor 488-conjugated goat anti-human IgG (Molecular Probes) and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes).

    Techniques: Flow Cytometry, Cytometry, Transfection, Staining, Incubation, Labeling, Fluorescence

    Apoptosis-modulating ϕ mutations alter μ1 distribution in cells. (A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P

    Journal: PLoS Pathogens

    Article Title: Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the ?1 ? Domain

    doi: 10.1371/journal.ppat.1000248

    Figure Lengend Snippet: Apoptosis-modulating ϕ mutations alter μ1 distribution in cells. (A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P

    Article Snippet: Alexa Fluor-conjugated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG secondary antibodies were purchased from Invitrogen.

    Techniques: Infection, Mutagenesis

    Immunofluorescence on cells expressing wild-type or chimeric rmGlu 6 or rmGlu 7 constructs. Cells were stained after fixation and permeabilization with MAB1/28-IgG followed by secondary antibody labelled with Alexa Fluor 647. In the top panel the predicted receptor topology is indicated relative to the membrane with the large extracellular domain at the top, with the mGlu 6 part in grey and mGlu 7 in black. Shown are representative overlaid images of immunostain (green) and nuclear stain (blue) acquired at 20 × magnification using same exposure parameters for the different are shown.

    Journal: British Journal of Pharmacology

    Article Title: Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7

    doi: 10.1111/j.1476-5381.2012.02090.x

    Figure Lengend Snippet: Immunofluorescence on cells expressing wild-type or chimeric rmGlu 6 or rmGlu 7 constructs. Cells were stained after fixation and permeabilization with MAB1/28-IgG followed by secondary antibody labelled with Alexa Fluor 647. In the top panel the predicted receptor topology is indicated relative to the membrane with the large extracellular domain at the top, with the mGlu 6 part in grey and mGlu 7 in black. Shown are representative overlaid images of immunostain (green) and nuclear stain (blue) acquired at 20 × magnification using same exposure parameters for the different are shown.

    Article Snippet: FITC-rabbit anti-mouse IgG (H + L), rabbit anti-mouse IgG (H + L) specific antibody, Alexa Fluor 532 goat anti-mouse IgG (H + L), Alexa Fluor 647 goat anti-mouse IgG (H + L) and Alexa Fluor 647 goat anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA, USA) and the anti-mGlu6 rabbit polyclonal antibody ab90866 was purchased from Abcam (Cambridge, UK).

    Techniques: Immunofluorescence, Expressing, Construct, Staining