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  • 99
    Thermo Fisher alexa fluor 594
    EWI-2 is downregulated from the surface of infected cells. ( A ) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4 + T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [ 50 , 51 ]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. ( B ) Cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, stained with DAPI (shown in cyan) and <t>Alexa</t> Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. ( C ) Cells were prepared as in ( B ) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated fluorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantified. Error bars = SD. p -values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by flow cytometry. ( D ) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates defining EWI-2 high and EWI-2 low cells are shown. ( E ) Data represent the percentage of uninfected and infected cells that fell into the EWI-2 high gate shown in ( d ) from 3 independent biological replicates, averaged across two technical replicates within each. ( F ) EWI-2 surface expression was measured by mean fluorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate.
    Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 phalloidin
    The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing <t>Alexa</t> Fluor <t>594–phalloidin</t> decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of β-catenin in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.
    Alexa Fluor 594 Phalloidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2894 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 594 conjugated secondary antibodies
    Cyclopamine targets both PGCs and somatic cells to effect PGC mislocalization (A) Schematic representation of the transplantation procedure used to differentiate cyclopamine action on PGCs and somatic tissues. Donor embryos were injected at the one-cell stage with EGFP-nanos1- 3’UTR mRNA to selectively label PGCs and <t>Alexa</t> Fluor 594-dextran to label all donor cells, and one to three PGCs were transplanted from shield-stage (6 hpf) donors into unlabeled sphere-stage (4 hpf) hosts. Donor and host embryos were treated with 100 μM cyclopamine or an ethanol vehicle control prior to transplantation, and cultured until 24 hpf in cyclopamine-free medium. EGFP-expressing PGCs in the host embryos were then scored at 24 hpf for their localization to the presumptive gonad site. (B-E) Bright-field images, (B’-E’) EGFP fluorescence images, and (B”-E”) Alexa <t>Fluor</t> 594 fluorescence images of representative host embryos resulting from the following donor/host treatment regimens: (B) ethanol-treated donor and host; (C) cyclopamine-treated donor and host; (D) cyclopamine-treated donor and ethanol-treated host; and (E) ethanol-treated donor and cyclopamine-treated host. (F) Quantification of PGC mislocalization, scoring at least 100 transplanted PGCs for each donor/host condition per experiment. Data represent the average of three or more experiments, with error bars representing the standard error of the mean. (G-L) Distribution of sdf1a transcripts in ethanol-(G-I) and cyclopamine-treated (J-L) embryos from gastrulation to mid-somitogenesis. Embryos at the shield (G and J), 3-somite (H and K), and 15-somite (I and L) stages are shown. Scale bars: 200 μm.
    Alexa Fluor 594 Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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 594 goat anti mouse igg
    GCaMP6-based Ca 2+ imaging in MagR-expressing 293 cells . Fibroblast HEK293A cells were transfected with either a plasmid in which MagR and GCaMP6 were linked by P2A sequence (GCaMP6-P2A-MagR), or co-transfected with MagR-mCherry and GCaMP6. On the next day, cells were subjected to magnetic field stimulation while Ca 2+ changes were monitored by epifluorescence imaging. ATP was applied at the end of experiments to verify the viability of transfected cells and the ability of GCaMP6 to monitor changes in intracellular Ca 2+ . (A) Representative images showing the fluorescent intensity of HEK293 cells before (Field off) and during (Field on) magnetic stimulation, and a few seconds after applying ATP (ATP). Scale bar: 20 μm. (B–D) Quantification of the fluorescent intensity changes (ΔF/F 0 ) in 293 cells transfected with the indicated constructs over time upon 1.0 mT magnetic field stimulation. Magnetic stimulation was indicated by blue bars above the curve, ATP was applied after switching off the magnetic field (arrows). The gradual decline of ΔF/F 0 over time was due to photo bleaching. “n” indicates the number of cells recorded. (E,F) Stimulation-response curves. Cell responses were determined under different magnetic field strengths. ΔF/F 0 at 27 s. after switching on of magnetic field was plotted against field strengths. Linear regression models show no correlation between MS field strengths and ΔF/F 0 (For 1E, y = −0.11x ( r 2 = 0.46; for 1F, y = −0.09x ( r 2 = 0.089). The slopes of both curves were not significantly different from zero [ F (1, 4) = 3.36, p = 0.14, and F (1, 4) = 0.39, p = 0.57, respectively]. No changes were observed for magnetic field stimulation up to 1.0 mT. In this and all other figures, data are presented in Mean ± SEM. (G) Western blot showing the expression of MagR protein in transfected cells. HEK293 cells were transfected with MagR-mCherry. A monoclonal antibody specific for MagR was used to detect MagR expression. Purified recombinant MagR is used as a positive control and lysates from cells not transfected or transfected with mCherry alone were used as negative controls. (H) Immunostaining showing co-expression of MagR and GCaMP6s proteins in transfected cells. HEK293 cells transfected with GCaMP6s (upper panels) or GCaMP6-P2A-MagR (lower panels), and immunostained with a mouse monoclonal anti-MagR antibody, followed by <t>Alexa</t> <t>Fluor®594</t> goat anti-mouse <t>IgG</t> secondary antibody (excitation wavelength 594 nm). GCaMP6 was excited at 488 nm. The cells were also nuclear stained with DAPI (405 nm). Merge views show co-localization of MagR and GCaMP6s. Scale bar = 10 μm.
    Alexa Fluor 594 Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2483 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
    GCaMP6-based Ca 2+ imaging in MagR-expressing 293 cells . Fibroblast HEK293A cells were transfected with either a plasmid in which MagR and GCaMP6 were linked by P2A sequence (GCaMP6-P2A-MagR), or co-transfected with MagR-mCherry and GCaMP6. On the next day, cells were subjected to magnetic field stimulation while Ca 2+ changes were monitored by epifluorescence imaging. ATP was applied at the end of experiments to verify the viability of transfected cells and the ability of GCaMP6 to monitor changes in intracellular Ca 2+ . (A) Representative images showing the fluorescent intensity of HEK293 cells before (Field off) and during (Field on) magnetic stimulation, and a few seconds after applying ATP (ATP). Scale bar: 20 μm. (B–D) Quantification of the fluorescent intensity changes (ΔF/F 0 ) in 293 cells transfected with the indicated constructs over time upon 1.0 mT magnetic field stimulation. Magnetic stimulation was indicated by blue bars above the curve, ATP was applied after switching off the magnetic field (arrows). The gradual decline of ΔF/F 0 over time was due to photo bleaching. “n” indicates the number of cells recorded. (E,F) Stimulation-response curves. Cell responses were determined under different magnetic field strengths. ΔF/F 0 at 27 s. after switching on of magnetic field was plotted against field strengths. Linear regression models show no correlation between MS field strengths and ΔF/F 0 (For 1E, y = −0.11x ( r 2 = 0.46; for 1F, y = −0.09x ( r 2 = 0.089). The slopes of both curves were not significantly different from zero [ F (1, 4) = 3.36, p = 0.14, and F (1, 4) = 0.39, p = 0.57, respectively]. No changes were observed for magnetic field stimulation up to 1.0 mT. In this and all other figures, data are presented in Mean ± SEM. (G) Western blot showing the expression of MagR protein in transfected cells. HEK293 cells were transfected with MagR-mCherry. A monoclonal antibody specific for MagR was used to detect MagR expression. Purified recombinant MagR is used as a positive control and lysates from cells not transfected or transfected with mCherry alone were used as negative controls. (H) Immunostaining showing co-expression of MagR and GCaMP6s proteins in transfected cells. HEK293 cells transfected with GCaMP6s (upper panels) or GCaMP6-P2A-MagR (lower panels), and immunostained with a mouse monoclonal anti-MagR antibody, followed by <t>Alexa</t> <t>Fluor®594</t> goat anti-mouse <t>IgG</t> secondary antibody (excitation wavelength 594 nm). GCaMP6 was excited at 488 nm. The cells were also nuclear stained with DAPI (405 nm). Merge views show co-localization of MagR and GCaMP6s. Scale bar = 10 μm.
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher click it edu alexa fluor 594 imaging kit
    Demonstration of IFI16 mediated H3K9me3 dependent recruitment of Heterochromatin Protein 1-α (HP1α). ( A ) TIME cells were infected with <t>EdU-KSHV</t> and stained using the Click-iT EdU <t>Alexa</t> <t>Fluor</t> 594 Imaging Kit (red). Subsequently, IFA was performed against HP1α (green). Colocalization of green (IFA) with red (EdU-KSHV genome) resulting in yellow indicates recruitment of HP1α on the KSHV genome (enlarged image, white arrows). ( B ) HP1α was KD in BCBL-1 cells by shRNA for 96 hr and KD efficiency assessed by qRT PCR using primers specific for HP1α as well as HP1β and HP1γ. ( C ) WB to confirm efficient KD. ( D ) KSHV mRNA levels were assessed by qRT PCR after HP1α KD. IFI16 mRNA was also assessed to confirm that the effect is specific for the KSHV genome only. mRNA levels were normalized against β-tubulin mRNA and expressed as relative amounts compared to shC-treated cells. ( E ) TIME cells were either infected with EdU-KSHV or control KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( F ) IFI16 was KD in TIME cells using siIFI16. After 72 hr, cells were infected with EdU-KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( G ) HP1α ChIP was performed 48 hr of de novo infection of TIME cells previously KD of IFI16 for 72 hr. KSHV promoters pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L were tested by q-PCR. ChIP efficiencies have been normalized to input chromatin and are represented as relative to shC control. Data shown are averages of the results of at least three experiments ± SD (*, p
    Click It Edu Alexa Fluor 594 Imaging Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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 goat anti rabbit alexa fluor 594
    SMase treatment of MDBK cells reduces sphingomyelin levels. Confluent MDBK monolayers were treated with DMEM (A) or 10 U/ml S. aureus SMase (B) for 45 min at 37°C. Cells were fixed with 6% paraformaldehyde and incubated with 0.5 μM lysenin for 2 h. Rabbit polyclonal antibody to lysenin was added and then detected with <t>Alexa</t> Fluor 594-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with DAPI. Cells were visualized by fluorescence microscopy. Magnification, ×40. ImageJ software was used to measure the mean fluorescence intensity from five equal areas per sample, each containing ∼150 to 250 cells. Results are representative of two independent experiments. The P value was determined using Student’s t test. (*, P
    Goat Anti Rabbit Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 conjugated goat anti mouse igg
    Visualization of ACR-YFP and calnexin by immunofluorescence. Axenically grown acr − /ACR-YFP cells were harvested in exponential phase and triple stained with (i) a polyclonal rabbit-anti-GFP antibody, followed by FITC conjugated donkey anti-rabbit <t>IgG;</t> (ii) a monoclonal mouse-anti-calnexin antibody ( 24 ) followed by <t>Alexa</t> Fluor® 594 conjugated goat anti-mouse IgG; and (iii) DAPI to detect ACR-YFP, calnexin, and DNA, respectively. Cells were photographed through the UV, TRITC, and FITC filter sets of a Leica DMLB2 fluorescence microscope. The merged image was prepared with the Qcapture Pro camera software. Scale bar , 10 μm.
    Alexa Fluor 594 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1027 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 conjugate
    NSF inmunodetection during meiotic maturation in wild type and hyh MII oocytes. ( a ) NSF was detected by indirect immunoflurescence in wild type and mutant homozygous (hyh) GV-intact oocytes (GV) and MII oocytes (MII). Red indicates positive staining for primary NSF antibody detected by a secondary antibody conjugated to <t>Alexa</t> Fluor 594; blue indicates DNA, labeled with Hoechst 3342; DIC shows differential interference contrast (DIC) images. Right column in each panel shows the fluorescence intensity profiles for NSF. Fluorescence intensities were measured along equatorial dashed yellow lines traced in each oocyte. The intensity of NSF is indicated by red lines. Scale bar: 20 μm. ( b ) Histogram showing fluorescence intensity quantification (A.U.: arbitrary units) for wild type GV and MII oocytes compared to hyh ones. ***p ≤ 0.001 (Student’s t-test).
    Alexa Fluor 594 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin alexa fluor 594 conjugate
    Confocal microscopic images of HEK 293T cells displaying scFv. HEK 293T cells transfected with a plasmid directing surface expression of anti-CD22 scFv were grown on coverslips. ( A – C ) Transfected cells were fixed with DAPI nuclear staining ( A ) followed by detection with biotinylated CD22-Fc ( B ), and anti-c-myc antibody ( C ) followed by <t>Steptavidin-Alexa</t> <t>Fluor-594</t> (red) and anti-mouse IgG-Alexa Fluor-488 (green). ( D ) Merged staining patterns (yellow) are shown. (Scale bar: 25 μm.)
    Streptavidin Alexa Fluor 594 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 731 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
    Confocal microscopic images of HEK 293T cells displaying scFv. HEK 293T cells transfected with a plasmid directing surface expression of anti-CD22 scFv were grown on coverslips. ( A – C ) Transfected cells were fixed with DAPI nuclear staining ( A ) followed by detection with biotinylated CD22-Fc ( B ), and anti-c-myc antibody ( C ) followed by <t>Steptavidin-Alexa</t> <t>Fluor-594</t> (red) and anti-mouse IgG-Alexa Fluor-488 (green). ( D ) Merged staining patterns (yellow) are shown. (Scale bar: 25 μm.)
    Goat Anti Rabbit Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher donkey anti rabbit igg h l highly cross adsorbed secondary antibody
    Confocal microscopic images of HEK 293T cells displaying scFv. HEK 293T cells transfected with a plasmid directing surface expression of anti-CD22 scFv were grown on coverslips. ( A – C ) Transfected cells were fixed with DAPI nuclear staining ( A ) followed by detection with biotinylated CD22-Fc ( B ), and anti-c-myc antibody ( C ) followed by <t>Steptavidin-Alexa</t> <t>Fluor-594</t> (red) and anti-mouse IgG-Alexa Fluor-488 (green). ( D ) Merged staining patterns (yellow) are shown. (Scale bar: 25 μm.)
    Donkey Anti Rabbit Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse alexa fluor 594
    NF-κB is involved in hypoxia-induced progression. ( a ) Cells were incubated under normoxic (N) or hypoxic (H) conditions, followed by isolation of nuclear protein extracts 24 hr later. DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. Specific (NF-κB) and unspecific shifts (UNSP) are marked. Competition with a tenfold excess of unlabeled oligonucleotide (10×) served as a control for specificity of binding. ( b ) Cells were transfected with siRNA oligonucleotides encoding a nonsense (NS) sequence or a specific sequence for inhibition of c-Rel (sicRel). Three days later, proteins were prepared, and the expression of c-Rel was evaluated by Western blot analysis. β-Actin served as a control for equal loading. The bar indicates 50 µm. ( c ) Three days after transfection with nonsense or c-Rel siRNA, the confluent cell layers were scratched, and then, they were cultured under hypoxic conditions. Closure of the wounded region was monitored 6 and 24 hr after scratching by microscopy and documented by photographs. The bar indicates 100 µm. ( d ) Likewise, the expression of EMT-related proteins Slug and Vimentin was detected by double immunofluorescence staining of cells exposed to hypoxia for 48 hr. DAPI counterstaining was used for detection of nuclei. Mouse anti-CD44 <t>mAb/Alexa</t> <t>Fluor</t> 594 IgG (red) and rabbit polyclonal anti-CA IX/Alexa Fluor 488 IgG (green) were used as secondary antibodies. Tissue sections were analyzed under ×400 magnification using a Leica DMRB fluorescence microscope. The bar indicates 25 µm. ( e ) Western blot analysis of Twist2 expression was performed using protein extracts derived from siRNA-transfected cells cultured under normoxia or hypoxia for 3 days.
    Goat Anti Mouse Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l highly cross adsorbed secondary antibody
    NF-κB is involved in hypoxia-induced progression. ( a ) Cells were incubated under normoxic (N) or hypoxic (H) conditions, followed by isolation of nuclear protein extracts 24 hr later. DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. Specific (NF-κB) and unspecific shifts (UNSP) are marked. Competition with a tenfold excess of unlabeled oligonucleotide (10×) served as a control for specificity of binding. ( b ) Cells were transfected with siRNA oligonucleotides encoding a nonsense (NS) sequence or a specific sequence for inhibition of c-Rel (sicRel). Three days later, proteins were prepared, and the expression of c-Rel was evaluated by Western blot analysis. β-Actin served as a control for equal loading. The bar indicates 50 µm. ( c ) Three days after transfection with nonsense or c-Rel siRNA, the confluent cell layers were scratched, and then, they were cultured under hypoxic conditions. Closure of the wounded region was monitored 6 and 24 hr after scratching by microscopy and documented by photographs. The bar indicates 100 µm. ( d ) Likewise, the expression of EMT-related proteins Slug and Vimentin was detected by double immunofluorescence staining of cells exposed to hypoxia for 48 hr. DAPI counterstaining was used for detection of nuclei. Mouse anti-CD44 <t>mAb/Alexa</t> <t>Fluor</t> 594 IgG (red) and rabbit polyclonal anti-CA IX/Alexa Fluor 488 IgG (green) were used as secondary antibodies. Tissue sections were analyzed under ×400 magnification using a Leica DMRB fluorescence microscope. The bar indicates 25 µm. ( e ) Western blot analysis of Twist2 expression was performed using protein extracts derived from siRNA-transfected cells cultured under normoxia or hypoxia for 3 days.
    Goat Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 594 donkey anti rabbit igg
    Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation. COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel A . Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel B . Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an <t>Alexa</t> Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa <t>Fluor</t> 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.
    Alexa Fluor 594 Donkey Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 892 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg h l highly cross adsorbed secondary antibody
    Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation. COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel A . Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel B . Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an <t>Alexa</t> Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa <t>Fluor</t> 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.
    Goat Anti Rabbit Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno alexa fluor 594 affinipure donkey anti rabbit igg
    Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation. COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel A . Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel B . Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an <t>Alexa</t> Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa <t>Fluor</t> 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.
    Alexa Fluor 594 Affinipure Donkey Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 403 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gfap
    Mitigation of surgery-induced inflammation in mice by disabling or reducing <t>IL17A</t> signaling. Expression of IL-6 (A) and TGFβ (B) mRNA and protein in hippocampus was measured by qRT-PCR and western-blotting assay on postoperative day 3. (C) Expression of <t>GFAP</t> in hippocampus was measure by i mmunofluorescene assay (magnification ×400). (D) Histology of paraffin sections of hippocampus isolated from IgG2a control mice or mice with IL17A antibody ICV infusion ( H E magnification ×200 ) on day 3 after surgery. Data are represented as means ± SEM. * P
    Gfap, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    EWI-2 is downregulated from the surface of infected cells. ( A ) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4 + T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [ 50 , 51 ]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. ( B ) Cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, stained with DAPI (shown in cyan) and Alexa Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. ( C ) Cells were prepared as in ( B ) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated fluorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantified. Error bars = SD. p -values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by flow cytometry. ( D ) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates defining EWI-2 high and EWI-2 low cells are shown. ( E ) Data represent the percentage of uninfected and infected cells that fell into the EWI-2 high gate shown in ( d ) from 3 independent biological replicates, averaged across two technical replicates within each. ( F ) EWI-2 surface expression was measured by mean fluorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate.

    Journal: Viruses

    Article Title: EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse

    doi: 10.3390/v11121082

    Figure Lengend Snippet: EWI-2 is downregulated from the surface of infected cells. ( A ) Abundance of EWI-2 in mock-infected (grey) versus WT HIV-infected (yellow) CEM-T4 T cells or primary human CD4 + T cells. Experiments were conducted in triplicate and whole cell lysates subjected to Tandem Mass Tag (TMT)-based quantitative proteomics 48 h after infection (reanalysis of data from [ 50 , 51 ]). Seven (CEM-T4 T cells) or six (primary human CD4+ T cells) unique peptides were used for EWI-2 quantitation. Mean relative abundances (fraction of maximum TMT reporter ion intensity) shown. ( B ) Cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, stained with DAPI (shown in cyan) and Alexa Fluor 594-conjugated secondary antibody, and imaged. GFP signal (yellow) was used to identify infected cells, and EWI-2-associated signal is shown pseudocolored in magenta. Representative cells are shown. Scale bars = 10 µm. ( C ) Cells were prepared as in ( B ) and EWI-2 levels at the plasma membrane in infected (Inf) and uninfected (Uninf) cells were measured by manually selecting the plasma membrane at the midline of each cell and quantifying the mean EWI-2-associated fluorescence intensity. Fluorescence intensity of each cell was normalized to the average intensity value of uninfected cells within the same imaging set. Data shown are pooled from two to three biological replicates, each consisting of two technical replicates. Only non-contact sites were quantified. Error bars = SD. p -values are the result of a two-tailed non-parametric Mann-Whitney U test. (C-E) CEM2n cells were infected with NL-sfGI and surface-labeled for EWI-2, fixed, and stained with Alexa Fluor 647-conjugated secondary antibody, and analyzed by flow cytometry. ( D ) Representative histogram normalized to mode of the EWI-2 signal intensity at the cell surface for unstained controls (black outline), infected cells (yellow), and uninfected cells (cyan). The gates defining EWI-2 high and EWI-2 low cells are shown. ( E ) Data represent the percentage of uninfected and infected cells that fell into the EWI-2 high gate shown in ( d ) from 3 independent biological replicates, averaged across two technical replicates within each. ( F ) EWI-2 surface expression was measured by mean fluorescence intensity (MFI) of EWI-2-associated signal. In both panels, lines connect paired data points, i.e., infected cells and uninfected cells (within an infected tube) from the same biological replicate.

    Article Snippet: Zenon labeling of primary antibodies with either Alexa Fluor 488 or Alexa Fluor 594 was carried out using Zenon Labeling Kits according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA, Cat. #Z25002 and #Z25007).

    Techniques: Infection, Quantitation Assay, Labeling, Staining, Fluorescence, Imaging, Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Cytometry, Expressing

    EWI-2 co-accumulates with Gag at the HIV-1 VS in T cells. ( A ) CEM-SS cells infected with HIV-1 NL4-3 WT or ΔEnv were co cultured with uninfected CEM-SS target cells for 5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). The EWI-2-associated fluorescence intensity at cell–cell contacts either enriched with Gag (WT) or not Gag-enriched but identified by DIC (ΔEnv) was measured. This value was then divided by the sum of the EWI-2-associated fluorescence intensity on non-contact sites on the producer and target cell in each VS/contact to yield EWI-2 enrichment (i.e., the values shown here). The data quantified are from one biological replicate consisting of two technical replicates. Similar trends were observed in a second dataset; not shown. ( B ) Primary CD4 + T cells infected with NL-sfGI WT or NL-CI ΔEnv were co-cultured with uninfected target primary cells for 2 h and stained for EWI-2 (magenta) and Gag (yellow), followed by secondary pAbs (Alexa Fluor 647-conjugated for EWI-2, and either Alexa Fluor 594 or Alexa Fluor 488-conjugated for Gag in the case of WT and ΔEnv, respectively). Because different secondary antibodies were used for Gag in either condition, the scaling shown for that channel is not the same across the two conditions and was based on corresponding primary and uninfected controls done alongside each dataset. Enrichment of EWI-2 at Env-dependent (WT) or Env-independent (ΔEnv) infected-uninfected cell contacts was quantified as described in ( A ). The data quantified are pooled from two independent biological replicates, each consisting of two technical replicates. Scale bars = 10 µm. In both data plots, each data point represents one cell–cell contact site (as opposed to one cell). The dotted horizontal line indicates a theoretical fold enrichment value of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p -values are the result of two-tailed non-parametric Mann-Whitney U tests.

    Journal: Viruses

    Article Title: EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse

    doi: 10.3390/v11121082

    Figure Lengend Snippet: EWI-2 co-accumulates with Gag at the HIV-1 VS in T cells. ( A ) CEM-SS cells infected with HIV-1 NL4-3 WT or ΔEnv were co cultured with uninfected CEM-SS target cells for 5 h, and subsequently stained for surface EWI-2 (magenta) and Gag (yellow). The EWI-2-associated fluorescence intensity at cell–cell contacts either enriched with Gag (WT) or not Gag-enriched but identified by DIC (ΔEnv) was measured. This value was then divided by the sum of the EWI-2-associated fluorescence intensity on non-contact sites on the producer and target cell in each VS/contact to yield EWI-2 enrichment (i.e., the values shown here). The data quantified are from one biological replicate consisting of two technical replicates. Similar trends were observed in a second dataset; not shown. ( B ) Primary CD4 + T cells infected with NL-sfGI WT or NL-CI ΔEnv were co-cultured with uninfected target primary cells for 2 h and stained for EWI-2 (magenta) and Gag (yellow), followed by secondary pAbs (Alexa Fluor 647-conjugated for EWI-2, and either Alexa Fluor 594 or Alexa Fluor 488-conjugated for Gag in the case of WT and ΔEnv, respectively). Because different secondary antibodies were used for Gag in either condition, the scaling shown for that channel is not the same across the two conditions and was based on corresponding primary and uninfected controls done alongside each dataset. Enrichment of EWI-2 at Env-dependent (WT) or Env-independent (ΔEnv) infected-uninfected cell contacts was quantified as described in ( A ). The data quantified are pooled from two independent biological replicates, each consisting of two technical replicates. Scale bars = 10 µm. In both data plots, each data point represents one cell–cell contact site (as opposed to one cell). The dotted horizontal line indicates a theoretical fold enrichment value of 1, which indicates no enrichment. Error bars = standard deviation of the mean (SD). p -values are the result of two-tailed non-parametric Mann-Whitney U tests.

    Article Snippet: Zenon labeling of primary antibodies with either Alexa Fluor 488 or Alexa Fluor 594 was carried out using Zenon Labeling Kits according to the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA, Cat. #Z25002 and #Z25007).

    Techniques: Infection, Cell Culture, Staining, Fluorescence, Standard Deviation, Two Tailed Test, MANN-WHITNEY

    localisation of FLAG tagged PATs and PPTs in  Trypanosoma cruzi  epimastigotes by immunofluorescence assay. Control: wild type epimastigote; blue: hoechst staining of nucleus (n) and kinetoplast (k) DNA; red: PAT (arrow) and PPT labeling with AlexaFluor 594. Bars = 5 µm.

    Journal: Memórias do Instituto Oswaldo Cruz

    Article Title: Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

    doi: 10.1590/0074-02760180086

    Figure Lengend Snippet: localisation of FLAG tagged PATs and PPTs in Trypanosoma cruzi epimastigotes by immunofluorescence assay. Control: wild type epimastigote; blue: hoechst staining of nucleus (n) and kinetoplast (k) DNA; red: PAT (arrow) and PPT labeling with AlexaFluor 594. Bars = 5 µm.

    Article Snippet: After three washes in PBS, the samples were incubated in the same conditions with a secondary goat anti-mouse antibody coupled to AlexaFluor 594 (Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:600 in incubation buffer.

    Techniques: Immunofluorescence, Staining, Labeling

    The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing Alexa Fluor 594–phalloidin decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of β-catenin in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing Alexa Fluor 594–phalloidin decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of β-catenin in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.

    Article Snippet: F-actin was labeled with Alexa Fluor 594–phalloidin (Invitrogen), and biotin was labeled with antibiotin-FITC (Vector Laboratories).

    Techniques: Labeling, Derivative Assay

    CD317 knockdown leads to activation of Rac. (A) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown Caco-2 cells transiently expressing either dominant-negative myc-tagged Rac or dominant-negative myc-tagged cdc42 as indicated (x = transfected cells) or after incubation of cells with the ROCK inhibitor Y-27632 as indicated. (B) Immunoblot analysis of phosphorylated Ser19 of MLC (MLCP) in lysates from CD317 knockdown, CD317 rescue, and control Caco-2 cells. An immunoblot of α-tubulin was used as a loading control. (C) Results of pull-down assay for active Rac showing an immunoblot of Rac detected with an anti-Rac antibody. The left lane of each pair shows the total amount of Rac present in 10% of the lysate used in the assay. The right lane of each pair shows the active Rac isolated in the pull-down in each case. Lysates were from the indicated polarized Caco-2 cells. Lysates from PMA-treated cells were used as a positive control for the presence of active Rac. (D) Ezrin and F-actin localization in polarized control (top) and CD317 knockdown (bottom) Caco-2 cells. The left pair of images shows XY sections of the apical surface of the cell monolayer, and the remaining images represent XZ sections of the monolayer. The basal F-actin image (bottom left) shows an XY section of F-actin in the basal region of the polarized CD317 knockdown Caco-2 cells. (E) Immunoblot analysis of C-terminal Thr phosphorylation of ezrin in the indicated Caco-2 cell lysates. Immunoblot of total ezrin was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (B, C, and E) Molecular mass is indicated in kilodaltons. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: CD317 knockdown leads to activation of Rac. (A) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown Caco-2 cells transiently expressing either dominant-negative myc-tagged Rac or dominant-negative myc-tagged cdc42 as indicated (x = transfected cells) or after incubation of cells with the ROCK inhibitor Y-27632 as indicated. (B) Immunoblot analysis of phosphorylated Ser19 of MLC (MLCP) in lysates from CD317 knockdown, CD317 rescue, and control Caco-2 cells. An immunoblot of α-tubulin was used as a loading control. (C) Results of pull-down assay for active Rac showing an immunoblot of Rac detected with an anti-Rac antibody. The left lane of each pair shows the total amount of Rac present in 10% of the lysate used in the assay. The right lane of each pair shows the active Rac isolated in the pull-down in each case. Lysates were from the indicated polarized Caco-2 cells. Lysates from PMA-treated cells were used as a positive control for the presence of active Rac. (D) Ezrin and F-actin localization in polarized control (top) and CD317 knockdown (bottom) Caco-2 cells. The left pair of images shows XY sections of the apical surface of the cell monolayer, and the remaining images represent XZ sections of the monolayer. The basal F-actin image (bottom left) shows an XY section of F-actin in the basal region of the polarized CD317 knockdown Caco-2 cells. (E) Immunoblot analysis of C-terminal Thr phosphorylation of ezrin in the indicated Caco-2 cell lysates. Immunoblot of total ezrin was used as a loading control. Black lines indicate that intervening lanes have been spliced out. (B, C, and E) Molecular mass is indicated in kilodaltons. Bars, 10 µm.

    Article Snippet: F-actin was labeled with Alexa Fluor 594–phalloidin (Invitrogen), and biotin was labeled with antibiotin-FITC (Vector Laboratories).

    Techniques: Activation Assay, Expressing, Dominant Negative Mutation, Transfection, Incubation, Pull Down Assay, Isolation, Positive Control

    Localization of CD317 in polarized Caco-2 cells, its knockdown in Caco-2 cells, and the effect of knockdown on F-actin. (A) Localization of CD317 in polarized Caco-2 cells. The top row shows XY images of CD317 (detected with an anti-CD317 antibody) and F-actin (Alexa Fluor 594–phalloidin) localization as indicated. The second row shows an XZ section from the first row. The third row shows XY images of CD317 and β-catenin localization as indicated (β-catenin is a marker of lateral membranes). The bottom row shows an XZ section from the third row. Panels on the right show merged images of the left and middle panels, with DAPI-stained nuclei on the right in the second row. (B) Immunoblot analysis (using an anti-CD317 antibody) of lysates from Caco-2 cells stably expressing CD317 siRNA or control GFP siRNA as indicated. The bands representing higher molecular weight proteins in the control siRNA lane correspond to glycosylated CD317. An immunoblot of α-tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown cells (top) and control cells (bottom). Each image shows a colony of cells with DAPI-stained nuclei. The top panel is an XZ section from a field of CD317 knockdown cells. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: Localization of CD317 in polarized Caco-2 cells, its knockdown in Caco-2 cells, and the effect of knockdown on F-actin. (A) Localization of CD317 in polarized Caco-2 cells. The top row shows XY images of CD317 (detected with an anti-CD317 antibody) and F-actin (Alexa Fluor 594–phalloidin) localization as indicated. The second row shows an XZ section from the first row. The third row shows XY images of CD317 and β-catenin localization as indicated (β-catenin is a marker of lateral membranes). The bottom row shows an XZ section from the third row. Panels on the right show merged images of the left and middle panels, with DAPI-stained nuclei on the right in the second row. (B) Immunoblot analysis (using an anti-CD317 antibody) of lysates from Caco-2 cells stably expressing CD317 siRNA or control GFP siRNA as indicated. The bands representing higher molecular weight proteins in the control siRNA lane correspond to glycosylated CD317. An immunoblot of α-tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown cells (top) and control cells (bottom). Each image shows a colony of cells with DAPI-stained nuclei. The top panel is an XZ section from a field of CD317 knockdown cells. Bars, 10 µm.

    Article Snippet: F-actin was labeled with Alexa Fluor 594–phalloidin (Invitrogen), and biotin was labeled with antibiotin-FITC (Vector Laboratories).

    Techniques: Marker, Staining, Stable Transfection, Expressing, Molecular Weight

    Visualization of 3O-C 12 -HSL-FITC and F-actin in Caco-2 cells. (A) Caco-2 cell monolayers were treated with 1 µM C 12 -HSL-FITC (green) or 0.018% DMSO as diluent control for 1, 5, 20, or 60 min. Cells were fixed and stained with Alexa Fluor 594-conjugated phalloidin to detect F-actin (red) and DAPI nucleic acid stain (blue), and were analyzed by confocal laser scanning microscopy. The images are from one representative of at least three independent experiments. Image size is 67.6×67.6 µm and pixel size is 0.13 µm. (B) Measurement of co-localization, based on Pearson's coefficient. Columns show the mean ± standard errors (n = 10) based on three independent experiments. Significant differences (*) in mean for Pearson's coefficient compared with values for control groups as calculated by Student's t test.

    Journal: PLoS Pathogens

    Article Title: The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration

    doi: 10.1371/journal.ppat.1002953

    Figure Lengend Snippet: Visualization of 3O-C 12 -HSL-FITC and F-actin in Caco-2 cells. (A) Caco-2 cell monolayers were treated with 1 µM C 12 -HSL-FITC (green) or 0.018% DMSO as diluent control for 1, 5, 20, or 60 min. Cells were fixed and stained with Alexa Fluor 594-conjugated phalloidin to detect F-actin (red) and DAPI nucleic acid stain (blue), and were analyzed by confocal laser scanning microscopy. The images are from one representative of at least three independent experiments. Image size is 67.6×67.6 µm and pixel size is 0.13 µm. (B) Measurement of co-localization, based on Pearson's coefficient. Columns show the mean ± standard errors (n = 10) based on three independent experiments. Significant differences (*) in mean for Pearson's coefficient compared with values for control groups as calculated by Student's t test.

    Article Snippet: To detect F-actin, fixed and permeabilized cells were stained with Alexa Fluor 594-conjugated phalloidin (Molecular Probes Invitrogen), diluted 1∶40 in PBS from 200 units/ml methanol stock solution, for 45 min at 37°C in the moist dark chamber.

    Techniques: Staining, Confocal Laser Scanning Microscopy

    Cyclopamine targets both PGCs and somatic cells to effect PGC mislocalization (A) Schematic representation of the transplantation procedure used to differentiate cyclopamine action on PGCs and somatic tissues. Donor embryos were injected at the one-cell stage with EGFP-nanos1- 3’UTR mRNA to selectively label PGCs and Alexa Fluor 594-dextran to label all donor cells, and one to three PGCs were transplanted from shield-stage (6 hpf) donors into unlabeled sphere-stage (4 hpf) hosts. Donor and host embryos were treated with 100 μM cyclopamine or an ethanol vehicle control prior to transplantation, and cultured until 24 hpf in cyclopamine-free medium. EGFP-expressing PGCs in the host embryos were then scored at 24 hpf for their localization to the presumptive gonad site. (B-E) Bright-field images, (B’-E’) EGFP fluorescence images, and (B”-E”) Alexa Fluor 594 fluorescence images of representative host embryos resulting from the following donor/host treatment regimens: (B) ethanol-treated donor and host; (C) cyclopamine-treated donor and host; (D) cyclopamine-treated donor and ethanol-treated host; and (E) ethanol-treated donor and cyclopamine-treated host. (F) Quantification of PGC mislocalization, scoring at least 100 transplanted PGCs for each donor/host condition per experiment. Data represent the average of three or more experiments, with error bars representing the standard error of the mean. (G-L) Distribution of sdf1a transcripts in ethanol-(G-I) and cyclopamine-treated (J-L) embryos from gastrulation to mid-somitogenesis. Embryos at the shield (G and J), 3-somite (H and K), and 15-somite (I and L) stages are shown. Scale bars: 200 μm.

    Journal: Developmental biology

    Article Title: Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent

    doi: 10.1016/j.ydbio.2009.01.036

    Figure Lengend Snippet: Cyclopamine targets both PGCs and somatic cells to effect PGC mislocalization (A) Schematic representation of the transplantation procedure used to differentiate cyclopamine action on PGCs and somatic tissues. Donor embryos were injected at the one-cell stage with EGFP-nanos1- 3’UTR mRNA to selectively label PGCs and Alexa Fluor 594-dextran to label all donor cells, and one to three PGCs were transplanted from shield-stage (6 hpf) donors into unlabeled sphere-stage (4 hpf) hosts. Donor and host embryos were treated with 100 μM cyclopamine or an ethanol vehicle control prior to transplantation, and cultured until 24 hpf in cyclopamine-free medium. EGFP-expressing PGCs in the host embryos were then scored at 24 hpf for their localization to the presumptive gonad site. (B-E) Bright-field images, (B’-E’) EGFP fluorescence images, and (B”-E”) Alexa Fluor 594 fluorescence images of representative host embryos resulting from the following donor/host treatment regimens: (B) ethanol-treated donor and host; (C) cyclopamine-treated donor and host; (D) cyclopamine-treated donor and ethanol-treated host; and (E) ethanol-treated donor and cyclopamine-treated host. (F) Quantification of PGC mislocalization, scoring at least 100 transplanted PGCs for each donor/host condition per experiment. Data represent the average of three or more experiments, with error bars representing the standard error of the mean. (G-L) Distribution of sdf1a transcripts in ethanol-(G-I) and cyclopamine-treated (J-L) embryos from gastrulation to mid-somitogenesis. Embryos at the shield (G and J), 3-somite (H and K), and 15-somite (I and L) stages are shown. Scale bars: 200 μm.

    Article Snippet: Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen/Molecular Probes) were used at a 1:1,000 dilution.

    Techniques: Pyrolysis Gas Chromatography, Transplantation Assay, Injection, Cell Culture, Expressing, Fluorescence

    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

    GCaMP6-based Ca 2+ imaging in MagR-expressing 293 cells . Fibroblast HEK293A cells were transfected with either a plasmid in which MagR and GCaMP6 were linked by P2A sequence (GCaMP6-P2A-MagR), or co-transfected with MagR-mCherry and GCaMP6. On the next day, cells were subjected to magnetic field stimulation while Ca 2+ changes were monitored by epifluorescence imaging. ATP was applied at the end of experiments to verify the viability of transfected cells and the ability of GCaMP6 to monitor changes in intracellular Ca 2+ . (A) Representative images showing the fluorescent intensity of HEK293 cells before (Field off) and during (Field on) magnetic stimulation, and a few seconds after applying ATP (ATP). Scale bar: 20 μm. (B–D) Quantification of the fluorescent intensity changes (ΔF/F 0 ) in 293 cells transfected with the indicated constructs over time upon 1.0 mT magnetic field stimulation. Magnetic stimulation was indicated by blue bars above the curve, ATP was applied after switching off the magnetic field (arrows). The gradual decline of ΔF/F 0 over time was due to photo bleaching. “n” indicates the number of cells recorded. (E,F) Stimulation-response curves. Cell responses were determined under different magnetic field strengths. ΔF/F 0 at 27 s. after switching on of magnetic field was plotted against field strengths. Linear regression models show no correlation between MS field strengths and ΔF/F 0 (For 1E, y = −0.11x ( r 2 = 0.46; for 1F, y = −0.09x ( r 2 = 0.089). The slopes of both curves were not significantly different from zero [ F (1, 4) = 3.36, p = 0.14, and F (1, 4) = 0.39, p = 0.57, respectively]. No changes were observed for magnetic field stimulation up to 1.0 mT. In this and all other figures, data are presented in Mean ± SEM. (G) Western blot showing the expression of MagR protein in transfected cells. HEK293 cells were transfected with MagR-mCherry. A monoclonal antibody specific for MagR was used to detect MagR expression. Purified recombinant MagR is used as a positive control and lysates from cells not transfected or transfected with mCherry alone were used as negative controls. (H) Immunostaining showing co-expression of MagR and GCaMP6s proteins in transfected cells. HEK293 cells transfected with GCaMP6s (upper panels) or GCaMP6-P2A-MagR (lower panels), and immunostained with a mouse monoclonal anti-MagR antibody, followed by Alexa Fluor®594 goat anti-mouse IgG secondary antibody (excitation wavelength 594 nm). GCaMP6 was excited at 488 nm. The cells were also nuclear stained with DAPI (405 nm). Merge views show co-localization of MagR and GCaMP6s. Scale bar = 10 μm.

    Journal: Frontiers in Neural Circuits

    Article Title: MagR Alone Is Insufficient to Confer Cellular Calcium Responses to Magnetic Stimulation

    doi: 10.3389/fncir.2017.00011

    Figure Lengend Snippet: GCaMP6-based Ca 2+ imaging in MagR-expressing 293 cells . Fibroblast HEK293A cells were transfected with either a plasmid in which MagR and GCaMP6 were linked by P2A sequence (GCaMP6-P2A-MagR), or co-transfected with MagR-mCherry and GCaMP6. On the next day, cells were subjected to magnetic field stimulation while Ca 2+ changes were monitored by epifluorescence imaging. ATP was applied at the end of experiments to verify the viability of transfected cells and the ability of GCaMP6 to monitor changes in intracellular Ca 2+ . (A) Representative images showing the fluorescent intensity of HEK293 cells before (Field off) and during (Field on) magnetic stimulation, and a few seconds after applying ATP (ATP). Scale bar: 20 μm. (B–D) Quantification of the fluorescent intensity changes (ΔF/F 0 ) in 293 cells transfected with the indicated constructs over time upon 1.0 mT magnetic field stimulation. Magnetic stimulation was indicated by blue bars above the curve, ATP was applied after switching off the magnetic field (arrows). The gradual decline of ΔF/F 0 over time was due to photo bleaching. “n” indicates the number of cells recorded. (E,F) Stimulation-response curves. Cell responses were determined under different magnetic field strengths. ΔF/F 0 at 27 s. after switching on of magnetic field was plotted against field strengths. Linear regression models show no correlation between MS field strengths and ΔF/F 0 (For 1E, y = −0.11x ( r 2 = 0.46; for 1F, y = −0.09x ( r 2 = 0.089). The slopes of both curves were not significantly different from zero [ F (1, 4) = 3.36, p = 0.14, and F (1, 4) = 0.39, p = 0.57, respectively]. No changes were observed for magnetic field stimulation up to 1.0 mT. In this and all other figures, data are presented in Mean ± SEM. (G) Western blot showing the expression of MagR protein in transfected cells. HEK293 cells were transfected with MagR-mCherry. A monoclonal antibody specific for MagR was used to detect MagR expression. Purified recombinant MagR is used as a positive control and lysates from cells not transfected or transfected with mCherry alone were used as negative controls. (H) Immunostaining showing co-expression of MagR and GCaMP6s proteins in transfected cells. HEK293 cells transfected with GCaMP6s (upper panels) or GCaMP6-P2A-MagR (lower panels), and immunostained with a mouse monoclonal anti-MagR antibody, followed by Alexa Fluor®594 goat anti-mouse IgG secondary antibody (excitation wavelength 594 nm). GCaMP6 was excited at 488 nm. The cells were also nuclear stained with DAPI (405 nm). Merge views show co-localization of MagR and GCaMP6s. Scale bar = 10 μm.

    Article Snippet: Next day, the cells were rinsed 3 times in PBS, and exposed to Alexa Fluor®647 donkey anti-mouse IgG (1:500, Invitrogen, Carlsbad, CA) or Alexa Fluor®594 goat anti-mouse IgG (1:500, Invitrogen, Carlsbad, CA) secondary antibodies for 1 h in a dark chamber followed by counterstaining with 10 μg/ml DAPI for 10 min at room temperature.

    Techniques: Imaging, Expressing, Transfection, Plasmid Preparation, Sequencing, Construct, Mass Spectrometry, Western Blot, Purification, Recombinant, Positive Control, Immunostaining, Staining

    Demonstration of IFI16 mediated H3K9me3 dependent recruitment of Heterochromatin Protein 1-α (HP1α). ( A ) TIME cells were infected with EdU-KSHV and stained using the Click-iT EdU Alexa Fluor 594 Imaging Kit (red). Subsequently, IFA was performed against HP1α (green). Colocalization of green (IFA) with red (EdU-KSHV genome) resulting in yellow indicates recruitment of HP1α on the KSHV genome (enlarged image, white arrows). ( B ) HP1α was KD in BCBL-1 cells by shRNA for 96 hr and KD efficiency assessed by qRT PCR using primers specific for HP1α as well as HP1β and HP1γ. ( C ) WB to confirm efficient KD. ( D ) KSHV mRNA levels were assessed by qRT PCR after HP1α KD. IFI16 mRNA was also assessed to confirm that the effect is specific for the KSHV genome only. mRNA levels were normalized against β-tubulin mRNA and expressed as relative amounts compared to shC-treated cells. ( E ) TIME cells were either infected with EdU-KSHV or control KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( F ) IFI16 was KD in TIME cells using siIFI16. After 72 hr, cells were infected with EdU-KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( G ) HP1α ChIP was performed 48 hr of de novo infection of TIME cells previously KD of IFI16 for 72 hr. KSHV promoters pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L were tested by q-PCR. ChIP efficiencies have been normalized to input chromatin and are represented as relative to shC control. Data shown are averages of the results of at least three experiments ± SD (*, p

    Journal: eLife

    Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

    doi: 10.7554/eLife.49500

    Figure Lengend Snippet: Demonstration of IFI16 mediated H3K9me3 dependent recruitment of Heterochromatin Protein 1-α (HP1α). ( A ) TIME cells were infected with EdU-KSHV and stained using the Click-iT EdU Alexa Fluor 594 Imaging Kit (red). Subsequently, IFA was performed against HP1α (green). Colocalization of green (IFA) with red (EdU-KSHV genome) resulting in yellow indicates recruitment of HP1α on the KSHV genome (enlarged image, white arrows). ( B ) HP1α was KD in BCBL-1 cells by shRNA for 96 hr and KD efficiency assessed by qRT PCR using primers specific for HP1α as well as HP1β and HP1γ. ( C ) WB to confirm efficient KD. ( D ) KSHV mRNA levels were assessed by qRT PCR after HP1α KD. IFI16 mRNA was also assessed to confirm that the effect is specific for the KSHV genome only. mRNA levels were normalized against β-tubulin mRNA and expressed as relative amounts compared to shC-treated cells. ( E ) TIME cells were either infected with EdU-KSHV or control KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( F ) IFI16 was KD in TIME cells using siIFI16. After 72 hr, cells were infected with EdU-KSHV for 24 hr followed by EdU-KSHV genome pulldown using Click chemistry. The inputs and eluates were blotted for the presence of HP1α. ( G ) HP1α ChIP was performed 48 hr of de novo infection of TIME cells previously KD of IFI16 for 72 hr. KSHV promoters pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L were tested by q-PCR. ChIP efficiencies have been normalized to input chromatin and are represented as relative to shC control. Data shown are averages of the results of at least three experiments ± SD (*, p

    Article Snippet: To fluorescent stained EdU labeled viral genome, a CLICK chemistry-based reaction was performed using Click-iTTM EdU Alexa FluorTM 594 imaging kit (Invitrogen #C10339) following the manufacturer’s instructions.

    Techniques: Infection, Staining, Imaging, Immunofluorescence, shRNA, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    Assessment of the specificity of the PLA reaction. TIME cells were infected with EdU-KSHV for 24 hr and stained using the Click-iT EdU Alexa Fluor 594 Imaging Kit (red). Subsequently, PLA (green) was performed between IgG (rabbit) and IgG (mouse) to assess the specificity of the PLA reaction.

    Journal: eLife

    Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

    doi: 10.7554/eLife.49500

    Figure Lengend Snippet: Assessment of the specificity of the PLA reaction. TIME cells were infected with EdU-KSHV for 24 hr and stained using the Click-iT EdU Alexa Fluor 594 Imaging Kit (red). Subsequently, PLA (green) was performed between IgG (rabbit) and IgG (mouse) to assess the specificity of the PLA reaction.

    Article Snippet: To fluorescent stained EdU labeled viral genome, a CLICK chemistry-based reaction was performed using Click-iTTM EdU Alexa FluorTM 594 imaging kit (Invitrogen #C10339) following the manufacturer’s instructions.

    Techniques: Proximity Ligation Assay, Infection, Staining, Imaging

    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

    SMase treatment of MDBK cells reduces sphingomyelin levels. Confluent MDBK monolayers were treated with DMEM (A) or 10 U/ml S. aureus SMase (B) for 45 min at 37°C. Cells were fixed with 6% paraformaldehyde and incubated with 0.5 μM lysenin for 2 h. Rabbit polyclonal antibody to lysenin was added and then detected with Alexa Fluor 594-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with DAPI. Cells were visualized by fluorescence microscopy. Magnification, ×40. ImageJ software was used to measure the mean fluorescence intensity from five equal areas per sample, each containing ∼150 to 250 cells. Results are representative of two independent experiments. The P value was determined using Student’s t test. (*, P

    Journal: Journal of Virology

    Article Title: Role of Sphingomyelin in Alphaherpesvirus Entry

    doi: 10.1128/JVI.01547-18

    Figure Lengend Snippet: SMase treatment of MDBK cells reduces sphingomyelin levels. Confluent MDBK monolayers were treated with DMEM (A) or 10 U/ml S. aureus SMase (B) for 45 min at 37°C. Cells were fixed with 6% paraformaldehyde and incubated with 0.5 μM lysenin for 2 h. Rabbit polyclonal antibody to lysenin was added and then detected with Alexa Fluor 594-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with DAPI. Cells were visualized by fluorescence microscopy. Magnification, ×40. ImageJ software was used to measure the mean fluorescence intensity from five equal areas per sample, each containing ∼150 to 250 cells. Results are representative of two independent experiments. The P value was determined using Student’s t test. (*, P

    Article Snippet: Goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific, Grand Island, NY) was stored at 4°C and diluted at 1:1,000 in 1% ovalbumin (Sigma-Aldrich) in PBS immediately prior to use.

    Techniques: Incubation, Fluorescence, Microscopy, Software

    DNA double strand breaks investigated by γ-H2AX detection. Mouse L929 fibroblasts were cultured for 96 h on nanowire and control substrates. After fixation, primary antibodies against γ-H2AX and secondary Alexa Fluor-594 conjugated antibodies were used to detect γ-H2AX (red). Actin was labeled with FITC-labeled phalloidin (green) and DNA was stained using bisbenzimide (blue). Fewer and larger (multinuclear) cells could be seen on the long nanowire substrate (right panel), where most cell nuclei stained positive for γ-H2AX. Scale bars 20 μm.

    Journal: Small (Weinheim an Der Bergstrasse, Germany)

    Article Title: Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    doi: 10.1002/smll.201300644

    Figure Lengend Snippet: DNA double strand breaks investigated by γ-H2AX detection. Mouse L929 fibroblasts were cultured for 96 h on nanowire and control substrates. After fixation, primary antibodies against γ-H2AX and secondary Alexa Fluor-594 conjugated antibodies were used to detect γ-H2AX (red). Actin was labeled with FITC-labeled phalloidin (green) and DNA was stained using bisbenzimide (blue). Fewer and larger (multinuclear) cells could be seen on the long nanowire substrate (right panel), where most cell nuclei stained positive for γ-H2AX. Scale bars 20 μm.

    Article Snippet: The samples were washed with PBS and incubated for 90 min with Alexa Fluor 594-goat-anti-rabbit (Invitrogen, Carlsbad, USA) (10 μg/mL) and FITC-labelled phalloidin (Sigma-Aldrich, St. Louis, USA) (1.7 μg/μL) in PBS with 1% (v/v) Tween-20 and 1% (w/v) BSA at room temperature.

    Techniques: Cell Culture, Labeling, Staining

    Visualization of ACR-YFP and calnexin by immunofluorescence. Axenically grown acr − /ACR-YFP cells were harvested in exponential phase and triple stained with (i) a polyclonal rabbit-anti-GFP antibody, followed by FITC conjugated donkey anti-rabbit IgG; (ii) a monoclonal mouse-anti-calnexin antibody ( 24 ) followed by Alexa Fluor® 594 conjugated goat anti-mouse IgG; and (iii) DAPI to detect ACR-YFP, calnexin, and DNA, respectively. Cells were photographed through the UV, TRITC, and FITC filter sets of a Leica DMLB2 fluorescence microscope. The merged image was prepared with the Qcapture Pro camera software. Scale bar , 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Functional Dissection of Adenylate Cyclase R, an Inducer of Spore Encapsulation *

    doi: 10.1074/jbc.M110.156380

    Figure Lengend Snippet: Visualization of ACR-YFP and calnexin by immunofluorescence. Axenically grown acr − /ACR-YFP cells were harvested in exponential phase and triple stained with (i) a polyclonal rabbit-anti-GFP antibody, followed by FITC conjugated donkey anti-rabbit IgG; (ii) a monoclonal mouse-anti-calnexin antibody ( 24 ) followed by Alexa Fluor® 594 conjugated goat anti-mouse IgG; and (iii) DAPI to detect ACR-YFP, calnexin, and DNA, respectively. Cells were photographed through the UV, TRITC, and FITC filter sets of a Leica DMLB2 fluorescence microscope. The merged image was prepared with the Qcapture Pro camera software. Scale bar , 10 μm.

    Article Snippet: After three washes with PBS, the cells were incubated for 2 h at 22 °C with 1:100 diluted FITC-conjugated donkey anti-rabbit IgG (Diagnostics Scotland, Edinburgh, Scotland) and 1:500 diluted Alexa Fluor® 594 conjugated goat anti-mouse IgG (Invitrogen) to detect the α-GFP and α-calnexin antibodies, respectively, and simultaneously counterstained with 0.1 μg/ml of the DNA stain DAPI (Invitrogen).

    Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy, Software

    NSF inmunodetection during meiotic maturation in wild type and hyh MII oocytes. ( a ) NSF was detected by indirect immunoflurescence in wild type and mutant homozygous (hyh) GV-intact oocytes (GV) and MII oocytes (MII). Red indicates positive staining for primary NSF antibody detected by a secondary antibody conjugated to Alexa Fluor 594; blue indicates DNA, labeled with Hoechst 3342; DIC shows differential interference contrast (DIC) images. Right column in each panel shows the fluorescence intensity profiles for NSF. Fluorescence intensities were measured along equatorial dashed yellow lines traced in each oocyte. The intensity of NSF is indicated by red lines. Scale bar: 20 μm. ( b ) Histogram showing fluorescence intensity quantification (A.U.: arbitrary units) for wild type GV and MII oocytes compared to hyh ones. ***p ≤ 0.001 (Student’s t-test).

    Journal: Scientific Reports

    Article Title: Pleiotropic effects of alpha-SNAP M105I mutation on oocyte biology: ultrastructural and cellular changes that adversely affect female fertility in mice

    doi: 10.1038/s41598-019-53574-8

    Figure Lengend Snippet: NSF inmunodetection during meiotic maturation in wild type and hyh MII oocytes. ( a ) NSF was detected by indirect immunoflurescence in wild type and mutant homozygous (hyh) GV-intact oocytes (GV) and MII oocytes (MII). Red indicates positive staining for primary NSF antibody detected by a secondary antibody conjugated to Alexa Fluor 594; blue indicates DNA, labeled with Hoechst 3342; DIC shows differential interference contrast (DIC) images. Right column in each panel shows the fluorescence intensity profiles for NSF. Fluorescence intensities were measured along equatorial dashed yellow lines traced in each oocyte. The intensity of NSF is indicated by red lines. Scale bar: 20 μm. ( b ) Histogram showing fluorescence intensity quantification (A.U.: arbitrary units) for wild type GV and MII oocytes compared to hyh ones. ***p ≤ 0.001 (Student’s t-test).

    Article Snippet: After washing, cells were incubated with the secondary antibody at RT for 1 h. The secondary antibodies used were: donkey anti-mouse, DyLight 488 conjugate (5 ng/μl, Jackson InmunoReasearch) to detect α SNAP; goat anti-rabbit, Alexa Fluor 594 conjugate (5 ng/μl, Thermo Fisher) to detect NSF.

    Techniques: Mutagenesis, Staining, Labeling, Fluorescence

    A , increased phosphorylation of histone H3 in neurons undergoing apoptosis. Neurons treated with or without DRB for 14 h were fixed and stained using Ser(P)-10-histone H3 polyclonal and MAP2 monoclonal antibodies. The staining was visualized using Alexa Fluor 594 and 488 secondary antibodies. The results show an increase in P-histone H3-positive neurons after KCl withdrawal ( center row ), and DRB inhibited this increase ( bottom row ). Magnification, ×63. B , coimmunostaining analysis did not show any colocalization of P-histone H3 and P-P70S6K. To determine whether the neurons that showed increased P-histone H3 show increased P-P70S6K, we performed coimmunostaining using the corresponding antibodies. Neurons were treated with or without DRB for 3 h, and, in a few wells, DRB was washed off, and neurons were replenished with medium deprived of KCl and cultured for an additional 3 h. The results showed an increase in P-histone H3- and P-P70S6K-positive neurons after KCl withdrawal ( arrowheads and arrows in the second row , respectively). We did not observe any colocalization of these two proteins in the neurons. Treatment with DRB inhibited the increase ( third row ), and removal of DRB led to an increase in the nuclear dispersion of P-P70S6K and appearance of P-histone H3-positive neurons. Magnification, ×40.

    Journal: The Journal of Biological Chemistry

    Article Title: Functional Role of RNA Polymerase II and P70 S6 Kinase in KCl Withdrawal-induced Cerebellar Granule Neuron Apoptosis *

    doi: 10.1074/jbc.M114.575225

    Figure Lengend Snippet: A , increased phosphorylation of histone H3 in neurons undergoing apoptosis. Neurons treated with or without DRB for 14 h were fixed and stained using Ser(P)-10-histone H3 polyclonal and MAP2 monoclonal antibodies. The staining was visualized using Alexa Fluor 594 and 488 secondary antibodies. The results show an increase in P-histone H3-positive neurons after KCl withdrawal ( center row ), and DRB inhibited this increase ( bottom row ). Magnification, ×63. B , coimmunostaining analysis did not show any colocalization of P-histone H3 and P-P70S6K. To determine whether the neurons that showed increased P-histone H3 show increased P-P70S6K, we performed coimmunostaining using the corresponding antibodies. Neurons were treated with or without DRB for 3 h, and, in a few wells, DRB was washed off, and neurons were replenished with medium deprived of KCl and cultured for an additional 3 h. The results showed an increase in P-histone H3- and P-P70S6K-positive neurons after KCl withdrawal ( arrowheads and arrows in the second row , respectively). We did not observe any colocalization of these two proteins in the neurons. Treatment with DRB inhibited the increase ( third row ), and removal of DRB led to an increase in the nuclear dispersion of P-P70S6K and appearance of P-histone H3-positive neurons. Magnification, ×40.

    Article Snippet: Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies were purchased from Invitrogen/Molecular Probes.

    Techniques: Staining, Cell Culture

    The coexpression of human IL-12p35 and p40 alters intracellular localization. A , HLtat cells were transfected with p35-FLAG ( A–D ), p40-FLAG ( E–H ), and the combinations of p35-FLAG and p40 ( I–L ) or p35 and p40-FLAG ( M–P ) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody ( A , E , I , and M ). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody ( B , F , J , and N ). The nuclei were visualized with DAPI ( C , G , K , and O ). The images were merged to show similar localization within the TGN ( D , H , L , and P ). B , increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.

    Journal: The Journal of Biological Chemistry

    Article Title: The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit

    doi: 10.1074/jbc.M112.436675

    Figure Lengend Snippet: The coexpression of human IL-12p35 and p40 alters intracellular localization. A , HLtat cells were transfected with p35-FLAG ( A–D ), p40-FLAG ( E–H ), and the combinations of p35-FLAG and p40 ( I–L ) or p35 and p40-FLAG ( M–P ) and fixed. FLAG-p35 and FLAG-p40 were visualized with anti-FLAG primary followed by Alexa-Fluor 488 secondary antibody ( A , E , I , and M ). The TGN was visualized with anti-TGN46 primary antibody followed by Alexa-Fluor 594 secondary antibody ( B , F , J , and N ). The nuclei were visualized with DAPI ( C , G , K , and O ). The images were merged to show similar localization within the TGN ( D , H , L , and P ). B , increase of glycosylated p35 in the presence of p40. The supernatants and cell lysates from HEK293 cells transfected with plasmids expressing the individual p35 or p40 subunits either alone or together were treated with Endo F or Endo H or left untreated. The samples were analyzed by Western immunoblot assay using the human IL-12p70 antibody. *, Endo H-resistant forms of p35 in the cell-associated fraction.

    Article Snippet: After 24 h, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, and incubated with mouse anti-FLAG (Sigma) and rabbit anti-TGN46 (Novus Biologicals, Littleton, CO) antibodies (at 1:1000 dilution each), followed by incubation with anti-mouse Alexa-Fluor 488 and anti-rabbit Alexa-Fluor 594 (Invitrogen) secondary antibodies.

    Techniques: Transfection, Expressing, Western Blot

    Confocal microscopic images of HEK 293T cells displaying scFv. HEK 293T cells transfected with a plasmid directing surface expression of anti-CD22 scFv were grown on coverslips. ( A – C ) Transfected cells were fixed with DAPI nuclear staining ( A ) followed by detection with biotinylated CD22-Fc ( B ), and anti-c-myc antibody ( C ) followed by Steptavidin-Alexa Fluor-594 (red) and anti-mouse IgG-Alexa Fluor-488 (green). ( D ) Merged staining patterns (yellow) are shown. (Scale bar: 25 μm.)

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

    Article Title: Isolation of anti-CD22 Fv with high affinity by Fv display on human cells

    doi: 10.1073/pnas.0603653103

    Figure Lengend Snippet: Confocal microscopic images of HEK 293T cells displaying scFv. HEK 293T cells transfected with a plasmid directing surface expression of anti-CD22 scFv were grown on coverslips. ( A – C ) Transfected cells were fixed with DAPI nuclear staining ( A ) followed by detection with biotinylated CD22-Fc ( B ), and anti-c-myc antibody ( C ) followed by Steptavidin-Alexa Fluor-594 (red) and anti-mouse IgG-Alexa Fluor-488 (green). ( D ) Merged staining patterns (yellow) are shown. (Scale bar: 25 μm.)

    Article Snippet: After incubation for 90 min at 25°C, cover slips were washed, incubated with Alexa Fluor-488-labeled goat anti-mouse IgG (1:500; Molecular Probes) and streptavidin-Alexa Fluor-594 conjugate (1:500; Molecular Probes) for 60 min at 25°C, and washed.

    Techniques: Transfection, Plasmid Preparation, Expressing, Staining

    NF-κB is involved in hypoxia-induced progression. ( a ) Cells were incubated under normoxic (N) or hypoxic (H) conditions, followed by isolation of nuclear protein extracts 24 hr later. DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. Specific (NF-κB) and unspecific shifts (UNSP) are marked. Competition with a tenfold excess of unlabeled oligonucleotide (10×) served as a control for specificity of binding. ( b ) Cells were transfected with siRNA oligonucleotides encoding a nonsense (NS) sequence or a specific sequence for inhibition of c-Rel (sicRel). Three days later, proteins were prepared, and the expression of c-Rel was evaluated by Western blot analysis. β-Actin served as a control for equal loading. The bar indicates 50 µm. ( c ) Three days after transfection with nonsense or c-Rel siRNA, the confluent cell layers were scratched, and then, they were cultured under hypoxic conditions. Closure of the wounded region was monitored 6 and 24 hr after scratching by microscopy and documented by photographs. The bar indicates 100 µm. ( d ) Likewise, the expression of EMT-related proteins Slug and Vimentin was detected by double immunofluorescence staining of cells exposed to hypoxia for 48 hr. DAPI counterstaining was used for detection of nuclei. Mouse anti-CD44 mAb/Alexa Fluor 594 IgG (red) and rabbit polyclonal anti-CA IX/Alexa Fluor 488 IgG (green) were used as secondary antibodies. Tissue sections were analyzed under ×400 magnification using a Leica DMRB fluorescence microscope. The bar indicates 25 µm. ( e ) Western blot analysis of Twist2 expression was performed using protein extracts derived from siRNA-transfected cells cultured under normoxia or hypoxia for 3 days.

    Journal: International Journal of Cancer. Journal International du Cancer

    Article Title: Triptolide reverses hypoxia-induced epithelial–mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation

    doi: 10.1002/ijc.28583

    Figure Lengend Snippet: NF-κB is involved in hypoxia-induced progression. ( a ) Cells were incubated under normoxic (N) or hypoxic (H) conditions, followed by isolation of nuclear protein extracts 24 hr later. DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. Specific (NF-κB) and unspecific shifts (UNSP) are marked. Competition with a tenfold excess of unlabeled oligonucleotide (10×) served as a control for specificity of binding. ( b ) Cells were transfected with siRNA oligonucleotides encoding a nonsense (NS) sequence or a specific sequence for inhibition of c-Rel (sicRel). Three days later, proteins were prepared, and the expression of c-Rel was evaluated by Western blot analysis. β-Actin served as a control for equal loading. The bar indicates 50 µm. ( c ) Three days after transfection with nonsense or c-Rel siRNA, the confluent cell layers were scratched, and then, they were cultured under hypoxic conditions. Closure of the wounded region was monitored 6 and 24 hr after scratching by microscopy and documented by photographs. The bar indicates 100 µm. ( d ) Likewise, the expression of EMT-related proteins Slug and Vimentin was detected by double immunofluorescence staining of cells exposed to hypoxia for 48 hr. DAPI counterstaining was used for detection of nuclei. Mouse anti-CD44 mAb/Alexa Fluor 594 IgG (red) and rabbit polyclonal anti-CA IX/Alexa Fluor 488 IgG (green) were used as secondary antibodies. Tissue sections were analyzed under ×400 magnification using a Leica DMRB fluorescence microscope. The bar indicates 25 µm. ( e ) Western blot analysis of Twist2 expression was performed using protein extracts derived from siRNA-transfected cells cultured under normoxia or hypoxia for 3 days.

    Article Snippet: Goat anti-rabbit Alexa Fluor 488 IgG and goat anti-mouse Alexa Fluor 594 (Invitrogen, Camarillo, CA) were used as secondary Abs.

    Techniques: Incubation, Isolation, Binding Assay, Labeling, Sequencing, Transfection, Inhibition, Expressing, Western Blot, Cell Culture, Microscopy, Double Immunofluorescence Staining, Fluorescence, Derivative Assay

    Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation. COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel A . Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel B . Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an Alexa Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa Fluor 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.

    Journal: PLoS ONE

    Article Title: IL-1? Stimulates COX-2 Dependent PGE2 Synthesis and CGRP Release in Rat Trigeminal Ganglia Cells

    doi: 10.1371/journal.pone.0017360

    Figure Lengend Snippet: Expression of COX-2 protein in trigeminal ganglia cells after IL-1β stimulation. COX-2 protein in cell culture homogenates was analyzed 6 hours after stimulation with IL-1β using Western blot (n = 3). A representative image is shown in panel A . Cell lysate of IL-1β treated TGC and the positive control (IFNy/LPS treated macrophages) showed a clear band at 70 kDa corresponding to COX-2 protein. Vehicle stimulation resulted in a faint COX-2 expression. The expression of COX-2 protein in cultured trigeminal ganglia cells exposed 6 hours to vehicle (10 ng/ml, upper panel B1-B3/B7-B9) or IL-1β (0.1 M PBS, lower panel B4-B9/B10-B12) is shown in fluorescent micrographs in panel B . Cells were stained with a mouse β-tubulin III antibody, indicative of neuronal cells (B1 and B4) or a mouse GFAP antibody, indicative of glial cells (B7 and B10), and a rabbit COX-2 antibody (B2, B5, B8, B11). The β-tubulin III and the GFAP antibodies were recognized by an Alexa Fluor 488 labeled secondary donkey anti-mouse antibody (green) and the COX-2 antibody was recognized by an Alexa Fluor 594 labeled secondary donkey anti-rabbit antibody (red). Double stained cells appear orange in B3, B6, B9 and B12. IL-1β caused a clear upregulation of COX-2 in neuronal and glial cells (lower panel) whereas a faint COX-2 expression could also be observed in control experiments (upper panel). The strongest induction of COX-2 was seen in bigger glial cells (40-100 µm) and neuronal cells.

    Article Snippet: The specimen was then washed 3×10 min in PBS and incubated for 90 min with Alexa Fluor 594 donkey anti-rabbit IgG (diluted 1∶600 in PBS; A-21207; Invitrogen, Karlsruhe, Germany) and Alexa Fluor 488 donkey anti-mouse IgG (diluted 1∶600 in PBS; A-21202; Invitrogen, Karlsruhe, Germany) +3% normal donkey serum and 0.3% Triton X in 0.1 M PBS at room temperature.

    Techniques: Expressing, Cell Culture, Western Blot, Positive Control, Staining, Labeling

    Mitigation of surgery-induced inflammation in mice by disabling or reducing IL17A signaling. Expression of IL-6 (A) and TGFβ (B) mRNA and protein in hippocampus was measured by qRT-PCR and western-blotting assay on postoperative day 3. (C) Expression of GFAP in hippocampus was measure by i mmunofluorescene assay (magnification ×400). (D) Histology of paraffin sections of hippocampus isolated from IgG2a control mice or mice with IL17A antibody ICV infusion ( H E magnification ×200 ) on day 3 after surgery. Data are represented as means ± SEM. * P

    Journal: PLoS ONE

    Article Title: Interleukin17A Promotes Postoperative Cognitive Dysfunction by Triggering β-Amyloid Accumulation via the Transforming Growth Factor-β (TGFβ)/Smad Signaling Pathway

    doi: 10.1371/journal.pone.0141596

    Figure Lengend Snippet: Mitigation of surgery-induced inflammation in mice by disabling or reducing IL17A signaling. Expression of IL-6 (A) and TGFβ (B) mRNA and protein in hippocampus was measured by qRT-PCR and western-blotting assay on postoperative day 3. (C) Expression of GFAP in hippocampus was measure by i mmunofluorescene assay (magnification ×400). (D) Histology of paraffin sections of hippocampus isolated from IgG2a control mice or mice with IL17A antibody ICV infusion ( H E magnification ×200 ) on day 3 after surgery. Data are represented as means ± SEM. * P

    Article Snippet: Antibodies for IL17A, IL6, TGFβ, GFAP, APP, and Aβ1–42 (Western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation

    Inhibition of TGFβ signaling blocks IL17A-stimulated co-expression of phospho-Smad2/3 (pSmad) with GFAP in mouse primary astrocytes. The administration of TGFβ receptor inhibitor (TβRI) reduced IL17A-induced pSmad 2/3 co-expression with GFAP in astrocytes. Double immunofluorescence staining showed that relative to IL17A (25 ng/ml) alone, pSmad 2/3 (red) co-localization with GFAP (green) in astrocytes following IL17A in combination with TβRI (5 μM) treatment was reduced. Magnification × 600.

    Journal: PLoS ONE

    Article Title: Interleukin17A Promotes Postoperative Cognitive Dysfunction by Triggering β-Amyloid Accumulation via the Transforming Growth Factor-β (TGFβ)/Smad Signaling Pathway

    doi: 10.1371/journal.pone.0141596

    Figure Lengend Snippet: Inhibition of TGFβ signaling blocks IL17A-stimulated co-expression of phospho-Smad2/3 (pSmad) with GFAP in mouse primary astrocytes. The administration of TGFβ receptor inhibitor (TβRI) reduced IL17A-induced pSmad 2/3 co-expression with GFAP in astrocytes. Double immunofluorescence staining showed that relative to IL17A (25 ng/ml) alone, pSmad 2/3 (red) co-localization with GFAP (green) in astrocytes following IL17A in combination with TβRI (5 μM) treatment was reduced. Magnification × 600.

    Article Snippet: Antibodies for IL17A, IL6, TGFβ, GFAP, APP, and Aβ1–42 (Western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Inhibition, Expressing, Double Immunofluorescence Staining