rabbit antibody alexa 488  (Thermo Fisher)


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    Name:
    F ab 2 Rabbit anti Goat IgG H L Cross Adsorbed Secondary Antibody
    Description:
    F ab 2 Rabbit anti Goat IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31109
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher rabbit antibody alexa 488
    Competitive binding of rDIII and EGF.  (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ®  488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM  125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
    F ab 2 Rabbit anti Goat IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    https://www.bioz.com/result/rabbit antibody alexa 488/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit antibody alexa 488 - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution"

    Article Title: Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200208145

    Competitive binding of rDIII and EGF.  (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ®  488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM  125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
    Figure Legend Snippet: Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Fluorescence, Concentration Assay

    Related Articles

    Staining:

    Article Title: Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation
    Article Snippet: The P31-43 sequence is LGQQQPFPPQQPY; and the P57-68 sequence is QLQPFPQPQLPY. .. Solutions were used in the following concentrations: P31-43-lissamine (liss), P31-43 CY3 and P57-68-liss at 20 micrograms/ml: unlabeled peptides were used as previously reported, at 70 micrograms/ml; EGF at 100 nanograms/ml; EGF-Alexa-488 at 20 nanograms/ml (Molecular Probes, San Giuliano Milanese, Italy); Dextran-Alexa488 (MW 10000) (Molecular Probes, San Giuliano Milanese, Italy) at 0.5 milligrams/ml; goat polyclonal antibody against EEA1 (C-15) at 2 micrograms/ml (Santa Cruz, DBA, Milan, Italy); mouse monoclonal antibody against LAMP2 (H4B4) at 2 micrograms/ml (Santa Cruz, DBA, Milan, Italy); secondary antibodies anti goat-Alexa-488 conjugated (Molecular Probes) for EEA1 staining at a ratio of 1∶100; and anti mouse-Alexa 488 conjugated (Molecular Probes) for LAMP2 staining at a ratio of 1∶100. ..

    Blocking Assay:

    Article Title: Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
    Article Snippet: .. V5 epitope-tagged proteins were detected using anti-V5 monoclonal antibody SV5-Pk1 and diluted 1:750 (Abcam catalog no. AB27671) in block solution at RT for 1 to 2 h. 3×HA-tagged proteins were detected using either an Alexa Fluor 488-conjugated anti-HA monoclonal antibody HA.11, diluted 1:250 (Nordic BioSite catalog no. 901509) in block solution or primary rabbit anti-HA monoclonal antibody C29F4, and diluted 1:1,600 (Cell Signaling catalog no. 3724S) in block solution at RT for 1 to 2 h. Primary antibodies were detected using Alexa Fluor 488-conjugated goat anti-mouse polyclonal antibody, diluted 1:800 (Life Technologies catalog no. A11029) in block solution at RT for 1 h or Alexa Fluor 488-conjugated goat anti-rabbit polyclonal antibody, and diluted 1:350 (Life Technologies catalog no. A11008) in block solution at RT for 1 h. Slides were mounted using VectaShield containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories catalog no. H-1200). .. Cells were viewed using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss GmbH), and images were processed using Zen lite 2012 (blue edition) version 1.1.2.0, ImageJ-Fiji version 1.51d, and Adobe Illustrator CC.

    Flow Cytometry:

    Article Title: Measles virus selectively blind to signaling lymphocyte activity molecule has oncolytic efficacy against nectin‐4‐expressing pancreatic cancer cells
    Article Snippet: Measles virus infection of MCF7 cells was detected by fluorescence of EGFP or by immunostaining using anti‐MV N protein mAb as previously described. .. Flow cytometry The expression of nectin‐4, SLAM, and CD46 was analyzed by flow cytometry according to previously reported methods., The following antibodies were used: anti‐human SLAM mAb (clone 7D4; Biolegend, San Diego, CA, USA); anti‐human CD46 mAb, anti‐nectin‐4 goat polyclonal antibody, mouse control IgG1, and goat control IgG (all from R & D Systems, Minneapolis, MN, USA); and Alexa 488‐conjugated anti‐mouse or anti‐goat antibody (Invitrogen). .. Cells were analyzed on a BD FACSVerse (BD Biosciences, San Diego, CA, USA).

    Cytometry:

    Article Title: Measles virus selectively blind to signaling lymphocyte activity molecule has oncolytic efficacy against nectin‐4‐expressing pancreatic cancer cells
    Article Snippet: Measles virus infection of MCF7 cells was detected by fluorescence of EGFP or by immunostaining using anti‐MV N protein mAb as previously described. .. Flow cytometry The expression of nectin‐4, SLAM, and CD46 was analyzed by flow cytometry according to previously reported methods., The following antibodies were used: anti‐human SLAM mAb (clone 7D4; Biolegend, San Diego, CA, USA); anti‐human CD46 mAb, anti‐nectin‐4 goat polyclonal antibody, mouse control IgG1, and goat control IgG (all from R & D Systems, Minneapolis, MN, USA); and Alexa 488‐conjugated anti‐mouse or anti‐goat antibody (Invitrogen). .. Cells were analyzed on a BD FACSVerse (BD Biosciences, San Diego, CA, USA).

    Expressing:

    Article Title: Measles virus selectively blind to signaling lymphocyte activity molecule has oncolytic efficacy against nectin‐4‐expressing pancreatic cancer cells
    Article Snippet: Measles virus infection of MCF7 cells was detected by fluorescence of EGFP or by immunostaining using anti‐MV N protein mAb as previously described. .. Flow cytometry The expression of nectin‐4, SLAM, and CD46 was analyzed by flow cytometry according to previously reported methods., The following antibodies were used: anti‐human SLAM mAb (clone 7D4; Biolegend, San Diego, CA, USA); anti‐human CD46 mAb, anti‐nectin‐4 goat polyclonal antibody, mouse control IgG1, and goat control IgG (all from R & D Systems, Minneapolis, MN, USA); and Alexa 488‐conjugated anti‐mouse or anti‐goat antibody (Invitrogen). .. Cells were analyzed on a BD FACSVerse (BD Biosciences, San Diego, CA, USA).

    Incubation:

    Article Title: A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells
    Article Snippet: .. After the pore formation step, cells were placed back on ice, washed twice with antibody incubation buffer (25 mM HEPES-KOH [pH 7.4], 110 mM potassium acetate, 2.5 mM magnesium acetate, 2 mM EGTA), and incubated with rabbit anti-Alexa-488 antibody (Life Technologies; antibody diluted in the antibody incubation buffer) on ice for 60 min. Unbound antibody was washed away, and cells were fixed with 3% paraformaldehyde (prepared in 25 mM HEPES-KOH [pH 7.4], 110 mM potassium acetate, and 2.5 mM magnesium acetate) at RT for 20 min, quenched for 10 min with 25 mM ammonium chloride in phosphate-buffered saline (PBS), and permeabilized with 0.5% Triton X-100 in PBS at RT for 5 min. .. Cells were blocked with 10% goat serum and stained with goat Alexa Fluor 594 (Alexa-594)-conjugated anti-rabbit antibodies (Life Technologies) and with 4′,6-diamidino-2-phenylindole (DAPI) to identify the nuclei.

    Fluorescence:

    Article Title: Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy
    Article Snippet: .. Samples were treated with 5 μg/ml rabbit anti-Alexa 488 antibody for 30 minutes on ice to quench surface Alexa 488 fluorescence, and subsequently fixed at room temperature with 3.4% (w/v) paraformaldehyde plus 0.025% (v/v) glutaraldehyde. .. Fixed cell samples were imaged with an Axio Observer Z1 inverted epifluorescent microscope (Zeiss, Oberkochen, Germany) equipped with a 63 X, 1.4 NA Plan-Apochromat objective (Zeiss), and a Zeiss 1.6 X internal optovar.

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    Thermo Fisher rabbit anti alexa 488 antibody
    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for <t>Alexa</t> 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.
    Rabbit Anti Alexa 488 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti alexa 488 antibody/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    96
    Thermo Fisher anti rabbit alexa 488
    Colocalization of EhAK1 with EhMyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 at the phagocytic cup during erythrophagocytosis. (A) Imaging of EhAK1, EhCaBP1 and EhCaBP3 during erythrophagocytosis. E. histolytica cells were incubated with RBC for 5 min at 37°C. The cells were then fixed and immunostained with anti-EhAK1 antibody followed by <t>Alexa-488.</t> F-actin was stained with TRITC-phalloidin and other indicated proteins were immunostained with respective antibodies and followed by Pacific blue-410. Arrowheads indicate phagocytic cups, asterisks mark just closed cups and star marks phagosome. Bar represents 5 µm. Quantitative analysis of fluorescent signals obtained by immunostaining of EhAK1, EhCaBP1 and EhCaBP3 from different locations in E. histolytica cells (N = 5) was done as described in Fig. 1D . (B) The incubation and labelling conditions were as described in Fig. 1 . Ehmyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 were immunostained with specific antibodies and visualized using Pacific blue-410 (blue). Colocalization analysis from five cells was done by using JACoP (ImageJ). The Pearson's coefficient (r) of EhAK1 with EhAK1, EhCaBP1, EhCaBP3, Ehmyosin 1B and EhC2PK from phagocytic cups are indicated. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.
    Anti Rabbit Alexa 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher goat anti rabbit alexa fluor 488
    Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse <t>Alexa-Fluor-488.</t> Cells were imaged on a widefield microscope.
    Goat Anti Rabbit Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit alexa fluor 488/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit alexa fluor 488 - by Bioz Stars, 2021-04
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    Image Search Results


    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Journal: Nature biotechnology

    Article Title: Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy

    doi: 10.1038/s41587-019-0073-7

    Figure Lengend Snippet: Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Article Snippet: Samples were treated with 5 μg/ml rabbit anti-Alexa 488 antibody for 30 minutes on ice to quench surface Alexa 488 fluorescence, and subsequently fixed at room temperature with 3.4% (w/v) paraformaldehyde plus 0.025% (v/v) glutaraldehyde.

    Techniques: Expressing, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

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

    Article Title: Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

    doi: 10.1073/pnas.2536800100

    Figure Lengend Snippet: Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Article Snippet: Stainings were visualized with Alexa 488-conjugated secondary anti-goat antibodies (Molecular Probes) and nuclei were counterstained with Hoechst 33342 (Molecular Probes).

    Techniques: Expressing, Mouse Assay, Injection, Staining, Immunofluorescence

    Colocalization of EhAK1 with EhMyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 at the phagocytic cup during erythrophagocytosis. (A) Imaging of EhAK1, EhCaBP1 and EhCaBP3 during erythrophagocytosis. E. histolytica cells were incubated with RBC for 5 min at 37°C. The cells were then fixed and immunostained with anti-EhAK1 antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin and other indicated proteins were immunostained with respective antibodies and followed by Pacific blue-410. Arrowheads indicate phagocytic cups, asterisks mark just closed cups and star marks phagosome. Bar represents 5 µm. Quantitative analysis of fluorescent signals obtained by immunostaining of EhAK1, EhCaBP1 and EhCaBP3 from different locations in E. histolytica cells (N = 5) was done as described in Fig. 1D . (B) The incubation and labelling conditions were as described in Fig. 1 . Ehmyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 were immunostained with specific antibodies and visualized using Pacific blue-410 (blue). Colocalization analysis from five cells was done by using JACoP (ImageJ). The Pearson's coefficient (r) of EhAK1 with EhAK1, EhCaBP1, EhCaBP3, Ehmyosin 1B and EhC2PK from phagocytic cups are indicated. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Journal: PLoS Pathogens

    Article Title: A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    doi: 10.1371/journal.ppat.1004411

    Figure Lengend Snippet: Colocalization of EhAK1 with EhMyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 at the phagocytic cup during erythrophagocytosis. (A) Imaging of EhAK1, EhCaBP1 and EhCaBP3 during erythrophagocytosis. E. histolytica cells were incubated with RBC for 5 min at 37°C. The cells were then fixed and immunostained with anti-EhAK1 antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin and other indicated proteins were immunostained with respective antibodies and followed by Pacific blue-410. Arrowheads indicate phagocytic cups, asterisks mark just closed cups and star marks phagosome. Bar represents 5 µm. Quantitative analysis of fluorescent signals obtained by immunostaining of EhAK1, EhCaBP1 and EhCaBP3 from different locations in E. histolytica cells (N = 5) was done as described in Fig. 1D . (B) The incubation and labelling conditions were as described in Fig. 1 . Ehmyosin 1B, EhC2PK, EhCaBP1 and EhCaBP3 were immunostained with specific antibodies and visualized using Pacific blue-410 (blue). Colocalization analysis from five cells was done by using JACoP (ImageJ). The Pearson's coefficient (r) of EhAK1 with EhAK1, EhCaBP1, EhCaBP3, Ehmyosin 1B and EhC2PK from phagocytic cups are indicated. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Article Snippet: Antibody dilutions used were: anti-EhAK1 at 1∶100, anti-EhCaBP1 at 1∶200, anti-EhCaBP3 at 1∶200, 1∶300 dilution of anti-rabbit Alexa 488, 556 and anti-mice Alexa556 (Molecular Probes).

    Techniques: Imaging, Incubation, Staining, Immunostaining

    EhAK1 is involved in the initiation of phagocytosis. (A) and (B) Fifty microgram of total cell lysate from indicated E. histolytica cell-lines was analyzed by western blotting to measure the levels of EhAK1. EhCaBP1 was used as an internal control. Cells (HM1:IMSS) were transfected with tet-inducible vector alone, antisense EhAK1 (AS) or sense EhAK1 (sense). Tetracycline (30 µg/ml) was added to induce gene expression in the +tet lanes. TOC is tet-o-CAT vector and AS is antisense. (C) Western blot analysis (as in panels, A and B) with cells carrying GFP-K85A construct of EhAK1 cloned in a constitutive vector. Cells were grown at the indicated concentration of G418. (D) RBC uptake assay was performed using the indicated cells grown with or without tet or G418. The experiments were carried out three times independently in triplicates. ANOVA test was used for statistical comparisons. (E) Quantitative determination of phagocytic cups observed in the indicated cell-lines was carried out by randomly selecting fifty cells in each experiment, and counting the number of phagocytic cups present in all these cells. (F) Cells were incubated with RBCs for the indicated time at 37°C. Cells were then fixed and stained for actin with TRITC-Phalloidin, or immunostained with anti-EhAK1 antibody followed by Alexa-488. The accumulation of actin at phagocytic cups is marked by solid arrowheads. Asterisks show attached RBCs at the site of phagocytosis. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005

    Journal: PLoS Pathogens

    Article Title: A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    doi: 10.1371/journal.ppat.1004411

    Figure Lengend Snippet: EhAK1 is involved in the initiation of phagocytosis. (A) and (B) Fifty microgram of total cell lysate from indicated E. histolytica cell-lines was analyzed by western blotting to measure the levels of EhAK1. EhCaBP1 was used as an internal control. Cells (HM1:IMSS) were transfected with tet-inducible vector alone, antisense EhAK1 (AS) or sense EhAK1 (sense). Tetracycline (30 µg/ml) was added to induce gene expression in the +tet lanes. TOC is tet-o-CAT vector and AS is antisense. (C) Western blot analysis (as in panels, A and B) with cells carrying GFP-K85A construct of EhAK1 cloned in a constitutive vector. Cells were grown at the indicated concentration of G418. (D) RBC uptake assay was performed using the indicated cells grown with or without tet or G418. The experiments were carried out three times independently in triplicates. ANOVA test was used for statistical comparisons. (E) Quantitative determination of phagocytic cups observed in the indicated cell-lines was carried out by randomly selecting fifty cells in each experiment, and counting the number of phagocytic cups present in all these cells. (F) Cells were incubated with RBCs for the indicated time at 37°C. Cells were then fixed and stained for actin with TRITC-Phalloidin, or immunostained with anti-EhAK1 antibody followed by Alexa-488. The accumulation of actin at phagocytic cups is marked by solid arrowheads. Asterisks show attached RBCs at the site of phagocytosis. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005

    Article Snippet: Antibody dilutions used were: anti-EhAK1 at 1∶100, anti-EhCaBP1 at 1∶200, anti-EhCaBP3 at 1∶200, 1∶300 dilution of anti-rabbit Alexa 488, 556 and anti-mice Alexa556 (Molecular Probes).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Construct, Clone Assay, Concentration Assay, Incubation, Staining

    EhAK1 is involved in phagocytosis. (A) Schematic representation of domain organization of EhAK1. EhAK1 is a 50 kDa protein with two domains, alpha kinase domain (38–267 amino acids) and SH3 domain (322–380 amino acids). The three different constructs used are shown. Lys 85 is the nucleoside triphosphate binding site, K85A mutant is the kinase dead mutant, KD-kinase domain alone. (B) Purified recombinant EhAK1 or K85A (2µg) was incubated in the presence of γ-32P-ATP, MgCl 2 and substrate (2µg) histone type (IIIS) at 30°C for 1 h. K85A mutant of EhAK1 exhibits no autophosphorylation and substrate phosphorylation activities. The products were analysed on SDS-PAGE and visualized in a phosphorimager. (C) Amoebic cells were stained for EhAK1 and EhTMKB1-9 (as membrane marker) using specific antibodies followed by Alexa488 and pacific blue-410, respectively. (D) Quantitative analysis of fluorescent signals obtained from panel (C). Intensity of immunostain (EhAK1, EhTMKB1-9) was measured at multiple locations in the membrane and cytosol and average relative intensity was computed by taking the signal from membrane as 100% for each marker separately. For analysis, five random regions were selected from membrane (blue) and cytosol (red) and average intensity was computed for each region. This was repeated for ten such cells (N = 10, bars represent standard error; scale bar, 5 µm). (E) Subcellular localization of EhAK1. E. histolytica whole cell lysate prepared from mid log phase cells, was fractionated biochemically in to cytoplasmic and membrane fractions as described in “Material and Methods”. Fifty micrograms of protein of indicated fraction was separated on SDS-PAGE, electrophoretically transferred to PVDF membranes and then immunostained with anti-EhAK1, anti-EhCaBP1 (cytoplasmic) and anti-EhTMK9 (membrane) antibodies as shown. (F) Imaging of EhAK1 and actin during erythrophagocytosis. E. histolytica cells were grown for 48 h and incubated with RBC for different times at 37°C. Immunostaining was performed using anti-EhAK1 antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin. Arrowheads indicate phagocytic cups, asterisks mark just closed cups (before cessation) and star marks phagosome. Bar represents 5 µm. (G) Quantitative analysis of fluorescent signals obtained by immunostaining of EhAK1 from different locations in E. histolytica cells (N = 5) as described in panel (D). *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Journal: PLoS Pathogens

    Article Title: A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    doi: 10.1371/journal.ppat.1004411

    Figure Lengend Snippet: EhAK1 is involved in phagocytosis. (A) Schematic representation of domain organization of EhAK1. EhAK1 is a 50 kDa protein with two domains, alpha kinase domain (38–267 amino acids) and SH3 domain (322–380 amino acids). The three different constructs used are shown. Lys 85 is the nucleoside triphosphate binding site, K85A mutant is the kinase dead mutant, KD-kinase domain alone. (B) Purified recombinant EhAK1 or K85A (2µg) was incubated in the presence of γ-32P-ATP, MgCl 2 and substrate (2µg) histone type (IIIS) at 30°C for 1 h. K85A mutant of EhAK1 exhibits no autophosphorylation and substrate phosphorylation activities. The products were analysed on SDS-PAGE and visualized in a phosphorimager. (C) Amoebic cells were stained for EhAK1 and EhTMKB1-9 (as membrane marker) using specific antibodies followed by Alexa488 and pacific blue-410, respectively. (D) Quantitative analysis of fluorescent signals obtained from panel (C). Intensity of immunostain (EhAK1, EhTMKB1-9) was measured at multiple locations in the membrane and cytosol and average relative intensity was computed by taking the signal from membrane as 100% for each marker separately. For analysis, five random regions were selected from membrane (blue) and cytosol (red) and average intensity was computed for each region. This was repeated for ten such cells (N = 10, bars represent standard error; scale bar, 5 µm). (E) Subcellular localization of EhAK1. E. histolytica whole cell lysate prepared from mid log phase cells, was fractionated biochemically in to cytoplasmic and membrane fractions as described in “Material and Methods”. Fifty micrograms of protein of indicated fraction was separated on SDS-PAGE, electrophoretically transferred to PVDF membranes and then immunostained with anti-EhAK1, anti-EhCaBP1 (cytoplasmic) and anti-EhTMK9 (membrane) antibodies as shown. (F) Imaging of EhAK1 and actin during erythrophagocytosis. E. histolytica cells were grown for 48 h and incubated with RBC for different times at 37°C. Immunostaining was performed using anti-EhAK1 antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin. Arrowheads indicate phagocytic cups, asterisks mark just closed cups (before cessation) and star marks phagosome. Bar represents 5 µm. (G) Quantitative analysis of fluorescent signals obtained by immunostaining of EhAK1 from different locations in E. histolytica cells (N = 5) as described in panel (D). *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Article Snippet: Antibody dilutions used were: anti-EhAK1 at 1∶100, anti-EhCaBP1 at 1∶200, anti-EhCaBP3 at 1∶200, 1∶300 dilution of anti-rabbit Alexa 488, 556 and anti-mice Alexa556 (Molecular Probes).

    Techniques: Construct, Binding Assay, Mutagenesis, Purification, Recombinant, Incubation, SDS Page, Staining, Marker, Imaging, Immunostaining

    In vivo localization of GFP-EhAK1 in phagocytosing cells. (A) and (C) Imaging of GFP-EhAK1 and GFP-KD and colocalization with actin during erythrophagocytosis. GFP-EhAK1 and GFP-KD expressing cells were grown for 48h with 30µg/ml G418 and incubated with RBC for different time at 37°C. Immunostaining was performed using anti-GFP antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin. EhCaBP1 was immunostained with specific antibodies and visualized using Pacific blue-410 (blue). Arrowheads indicate phagocytic cups, asterisks mark just closed cups and star marks phagosome. Bar represents 5 µm. (B) and (D) Quantitative analysis of fluorescent signals was done as in Fig. 1D . (E) Time lapse imaging of GFP-EhAK1. The montage shows a time series of GFP-EhAK1 expressing cells undergoing erythrophagocytosis where phagocytic cups are marked by arrowhead and just closed phagosomes by star. Bar represents 10 µm. Graph shows the intensity of GFP-EhAK1 (ROI) at a phagocytic cup every 3s. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Journal: PLoS Pathogens

    Article Title: A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    doi: 10.1371/journal.ppat.1004411

    Figure Lengend Snippet: In vivo localization of GFP-EhAK1 in phagocytosing cells. (A) and (C) Imaging of GFP-EhAK1 and GFP-KD and colocalization with actin during erythrophagocytosis. GFP-EhAK1 and GFP-KD expressing cells were grown for 48h with 30µg/ml G418 and incubated with RBC for different time at 37°C. Immunostaining was performed using anti-GFP antibody followed by Alexa-488. F-actin was stained with TRITC-phalloidin. EhCaBP1 was immunostained with specific antibodies and visualized using Pacific blue-410 (blue). Arrowheads indicate phagocytic cups, asterisks mark just closed cups and star marks phagosome. Bar represents 5 µm. (B) and (D) Quantitative analysis of fluorescent signals was done as in Fig. 1D . (E) Time lapse imaging of GFP-EhAK1. The montage shows a time series of GFP-EhAK1 expressing cells undergoing erythrophagocytosis where phagocytic cups are marked by arrowhead and just closed phagosomes by star. Bar represents 10 µm. Graph shows the intensity of GFP-EhAK1 (ROI) at a phagocytic cup every 3s. *p-value≤0.05, **p-value≤0.005, ***p-value≤0.0005.

    Article Snippet: Antibody dilutions used were: anti-EhAK1 at 1∶100, anti-EhCaBP1 at 1∶200, anti-EhCaBP3 at 1∶200, 1∶300 dilution of anti-rabbit Alexa 488, 556 and anti-mice Alexa556 (Molecular Probes).

    Techniques: In Vivo, Imaging, Expressing, Incubation, Immunostaining, Staining

    Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.

    Journal: eLife

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    doi: 10.7554/eLife.25716

    Figure Lengend Snippet: Anti-DDR1 mAbs inhibit collagen-induced DDR1 clustering. Cos-7 cells expressing DDR1 were incubated with 10 μg/ml collagen I for 10 minutes at 37°C, left unstimulated, or incubated with collagen I in the presence of function-blocking anti-DDR1 mAbs. Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488. Cells were imaged on a widefield microscope.

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); rabbit anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma); goat anti-mouse IgG Alexa-Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa-Fluor-488 (Invitrogen); goat anti-rabbit Alexa-Fluor-488 (Invitrogen), goat anti-mouse IgG2b Alexa-Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa-Fluor-555 (Invitrogen); and goat anti-rabbit IgG Alexa-Fluor-647 (Invitrogen).

    Techniques: Expressing, Incubation, Blocking Assay, Microscopy

    Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019

    Journal: eLife

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    doi: 10.7554/eLife.25716

    Figure Lengend Snippet: Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering. Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ), stimulated with 10 μg/ml collagen I ( B ), or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ), for 10 min at 37°C. Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab, and mounting. Cells were imaged using a widefield microscope. White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows. Scale bars, 30 μm (top rows) and 10 μm (bottom rows). DOI: http://dx.doi.org/10.7554/eLife.25716.019

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); rabbit anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma); goat anti-mouse IgG Alexa-Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa-Fluor-488 (Invitrogen); goat anti-rabbit Alexa-Fluor-488 (Invitrogen), goat anti-mouse IgG2b Alexa-Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa-Fluor-555 (Invitrogen); and goat anti-rabbit IgG Alexa-Fluor-647 (Invitrogen).

    Techniques: Blocking Assay, Incubation, Microscopy

    Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010

    Journal: eLife

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    doi: 10.7554/eLife.25716

    Figure Lengend Snippet: Cell surface expression of ectodomain-deletion constructs. ( A ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in Cos-7 cells. Cells were incubated on ice with mouse anti-Flag Ab, before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab. Cells were imaged using a widefield microscope. Scale bars, 20 µm. ( B ) N-terminally Flag-tagged ectodomain-deletion constructs (DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1) were transiently expressed in HEK293 cells. Cells were stained on ice with rabbit anti-Flag Ab, followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry. Filled g ray histograms , secondary Ab only; open histograms , anti-Flag. DOI: http://dx.doi.org/10.7554/eLife.25716.010

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); rabbit anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma); goat anti-mouse IgG Alexa-Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa-Fluor-488 (Invitrogen); goat anti-rabbit Alexa-Fluor-488 (Invitrogen), goat anti-mouse IgG2b Alexa-Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa-Fluor-555 (Invitrogen); and goat anti-rabbit IgG Alexa-Fluor-647 (Invitrogen).

    Techniques: Expressing, Construct, Incubation, Microscopy, Staining, Flow Cytometry, Cytometry

    Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020

    Journal: eLife

    Article Title: Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    doi: 10.7554/eLife.25716

    Figure Lengend Snippet: Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone. ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads (3 μm diameter) for 2 or 4 hr at 37°C. Control beads were coated with BSA and incubated for 2 hr at 37°C. Cells were incubated on ice with a mouse monoclonal Ab against the D DR1 ectodomain, before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. DDR1 (green) and pY513 (red) staining are shown in the merge image (right panel). ( B ) The ectodomain deletion construct DDR1b-ΔECD (which contains an N-terminal Flag tag) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells. Cells were stimulated with collagen-coated latex beads (3 μm diameter) for 2 hr at 37°C. Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs, followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab. Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555, and anti-rabbit Alexa-Fluor-647 secondary Abs. Flag (green) and pY513 (red) staining are shown in the merge image (right panel). BF, Brightfield. Scale bars, 10 μm. DOI: http://dx.doi.org/10.7554/eLife.25716.020

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); rabbit anti-goat Ig-horseradish peroxidase (Zymed Laboratories Inc., San Francisco, CA); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma); goat anti-mouse IgG Alexa-Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa-Fluor-488 (Invitrogen); goat anti-rabbit Alexa-Fluor-488 (Invitrogen), goat anti-mouse IgG2b Alexa-Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa-Fluor-555 (Invitrogen); and goat anti-rabbit IgG Alexa-Fluor-647 (Invitrogen).

    Techniques: Activation Assay, Ligand Binding Assay, Incubation, Staining, Construct, FLAG-tag