goat anti rabbit igg  (Thermo Fisher)


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    Name:
    Goat anti Rabbit IgG H L cross adsorbed
    Description:
    This product is a sample size 50 µG of Goat anti Rabbit IgG H L cross adsorbed horseradish peroxidase HRP Conjugate A16104 Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate is prepared from antibodies that have been adsorbed against bovine human and mouse IgG to minimize cross reactivity Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications The sensitivity of each lot of antibody is confirmed using ELISA The specificity of each lot of antibody is confirmed by isoelectric focusing IEF Applications Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications Host species The host species of theanti rabbit IgG H L horseradish peroxidase HRP Conjugate is goat Reactivity Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate detects rabbit IgG H L Product size 50 µg Suggested dilution The optimal working dilution should be determined empirically Dilutions of 1 500 1 5000 should be satisfactory for most immunohistochemical applications Dilutions of 1 200 1 5 000 should be satisfactory for most ELISA and western blot applications
    Catalog Number:
    A16104SAMPLE
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
    Applications:
    Build Your Own Immunoassay|Cell Analysis|Cellular Imaging|ELISA|Immunofluorescence (IF)|Immunofluorescence Staining & Detection|Protein Assays and Analysis|Protein Biology|Ready-To-Use Immunoassay|Secondary Detection
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    Structured Review

    Thermo Fisher goat anti rabbit igg
    Detection of chlamydial protein-specific antibodies in human sera by Western blotting. The chlamydial GST fusion proteins, including GST-CPAF (lane 1), GST-MOMP (lane 2), and GST-HSP60 (lane 3), were resolved on an SDS-polyacrylamide gel and transferred onto nitrocellulose membranes for measuring human antibody binding to the corresponding chlamydial proteins. The human sera were pooled from 10 patients and subjected to fivefold serial dilution (as indicated at the top of the figure). The primary antibody binding was visualized with an HRP-conjugated goat anti-human <t>IgG</t> and ECL. The GST-CPAF band was obviously detected after 1:312,500 dilution of the human sera while the GST-MOMP and GST-HSP60 were no longer or minimally detected at the same serum dilution. The leftmost panel is an image of the gel stained with brilliant blue dye for total amounts of protein loaded to each lane. Lane 0 was loaded with a prestained molecular mass marker.
    This product is a sample size 50 µG of Goat anti Rabbit IgG H L cross adsorbed horseradish peroxidase HRP Conjugate A16104 Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate is prepared from antibodies that have been adsorbed against bovine human and mouse IgG to minimize cross reactivity Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications The sensitivity of each lot of antibody is confirmed using ELISA The specificity of each lot of antibody is confirmed by isoelectric focusing IEF Applications Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate should be suitable for western blot ELISA immunohistochemistry and other standard immunoassay applications Host species The host species of theanti rabbit IgG H L horseradish peroxidase HRP Conjugate is goat Reactivity Goat Anti Rabbit IgG H L Antibody Horseradish Peroxidase HRP Conjugate detects rabbit IgG H L Product size 50 µg Suggested dilution The optimal working dilution should be determined empirically Dilutions of 1 500 1 5000 should be satisfactory for most immunohistochemical applications Dilutions of 1 200 1 5 000 should be satisfactory for most ELISA and western blot applications
    https://www.bioz.com/result/goat anti rabbit igg/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    Images

    1) Product Images from "Human Antibody Responses to a Chlamydia-Secreted Protease Factor "

    Article Title: Human Antibody Responses to a Chlamydia-Secreted Protease Factor

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.12.7164-7171.2004

    Detection of chlamydial protein-specific antibodies in human sera by Western blotting. The chlamydial GST fusion proteins, including GST-CPAF (lane 1), GST-MOMP (lane 2), and GST-HSP60 (lane 3), were resolved on an SDS-polyacrylamide gel and transferred onto nitrocellulose membranes for measuring human antibody binding to the corresponding chlamydial proteins. The human sera were pooled from 10 patients and subjected to fivefold serial dilution (as indicated at the top of the figure). The primary antibody binding was visualized with an HRP-conjugated goat anti-human IgG and ECL. The GST-CPAF band was obviously detected after 1:312,500 dilution of the human sera while the GST-MOMP and GST-HSP60 were no longer or minimally detected at the same serum dilution. The leftmost panel is an image of the gel stained with brilliant blue dye for total amounts of protein loaded to each lane. Lane 0 was loaded with a prestained molecular mass marker.
    Figure Legend Snippet: Detection of chlamydial protein-specific antibodies in human sera by Western blotting. The chlamydial GST fusion proteins, including GST-CPAF (lane 1), GST-MOMP (lane 2), and GST-HSP60 (lane 3), were resolved on an SDS-polyacrylamide gel and transferred onto nitrocellulose membranes for measuring human antibody binding to the corresponding chlamydial proteins. The human sera were pooled from 10 patients and subjected to fivefold serial dilution (as indicated at the top of the figure). The primary antibody binding was visualized with an HRP-conjugated goat anti-human IgG and ECL. The GST-CPAF band was obviously detected after 1:312,500 dilution of the human sera while the GST-MOMP and GST-HSP60 were no longer or minimally detected at the same serum dilution. The leftmost panel is an image of the gel stained with brilliant blue dye for total amounts of protein loaded to each lane. Lane 0 was loaded with a prestained molecular mass marker.

    Techniques Used: Western Blot, Binding Assay, Serial Dilution, Staining, Marker

    2) Product Images from "Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG"

    Article Title: Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00297-13

    Evaluation of the ability of purified IgM and IgG from immune sera to inhibit C. burnetii infection in vivo by comparing splenomegaly as well as bacterial burden and pathological changes in the spleen with those of normal mouse IgM and IgG controls at
    Figure Legend Snippet: Evaluation of the ability of purified IgM and IgG from immune sera to inhibit C. burnetii infection in vivo by comparing splenomegaly as well as bacterial burden and pathological changes in the spleen with those of normal mouse IgM and IgG controls at

    Techniques Used: Purification, Infection, In Vivo

    3) Product Images from "Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA"

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

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiy467

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

    Techniques Used: Binding Assay, Infection, Inhibition, Incubation

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

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

    4) Product Images from "Morphological and Functional Analyses of the Tight Junction in the Palatal Epithelium of Mouse"

    Article Title: Morphological and Functional Analyses of the Tight Junction in the Palatal Epithelium of Mouse

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.17006

    Immunohistochemistry of TJ constitutive proteins. The immunofluorescence of OCD ( A ), CLD-1 ( C ), and -4 ( D ) were localized between the upper layers of the stratum granulosum showing dot-like fluorescence (arrowheads). In addition, CLD-1 ( C ) and CLD-4 ( D ) showed network-like positive reaction from the lower layer of the stratum granulosum to the upper layer of the stratum spinosum. CLD-2 ( F ) and CLD-5 ( G ) were positive in the salivary glands and the endothelium of blood vessel respectively, but were negative in the palatal epithelium. Background staining levels were checked by omitting the primary antibodies ( B : rabbit anti-goat IgG labeled with Alexa Fluor 488, E : goat anti-rabbit IgG labeled with Alexa Fluor 488). SC: stratum corneum, SB: stratum basale, Blue: nuclei stained with Hoechst. Bars = 25 μm ( A, B, C, D, E ), 50 μm ( F ), 10 μm ( G ).
    Figure Legend Snippet: Immunohistochemistry of TJ constitutive proteins. The immunofluorescence of OCD ( A ), CLD-1 ( C ), and -4 ( D ) were localized between the upper layers of the stratum granulosum showing dot-like fluorescence (arrowheads). In addition, CLD-1 ( C ) and CLD-4 ( D ) showed network-like positive reaction from the lower layer of the stratum granulosum to the upper layer of the stratum spinosum. CLD-2 ( F ) and CLD-5 ( G ) were positive in the salivary glands and the endothelium of blood vessel respectively, but were negative in the palatal epithelium. Background staining levels were checked by omitting the primary antibodies ( B : rabbit anti-goat IgG labeled with Alexa Fluor 488, E : goat anti-rabbit IgG labeled with Alexa Fluor 488). SC: stratum corneum, SB: stratum basale, Blue: nuclei stained with Hoechst. Bars = 25 μm ( A, B, C, D, E ), 50 μm ( F ), 10 μm ( G ).

    Techniques Used: Immunohistochemistry, Immunofluorescence, Fluorescence, Staining, Labeling

    5) Product Images from "Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)"

    Article Title: Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

    Journal: bioRxiv

    doi: 10.1101/2020.03.12.988923

    Re-embedding enables Ex- d STORM. a , Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b , d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c , Zoom in on highlighted region in (b). d , Averaged cross-sectional profile of 9 microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in 2 cells from 1 expanded sample. e , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of the data analyzed in (c) with an average distance of 137.1 ± 10.1 nm (mean ± sd). f , Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g , Zoom in of the white boxed region in (f). h , Average intensity profile of 35 microtubule segments with a length between 1.1 – 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. d , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in (h) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). Scale bars, 2 µm (b,f), 500 nm (c,g).
    Figure Legend Snippet: Re-embedding enables Ex- d STORM. a , Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b , d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c , Zoom in on highlighted region in (b). d , Averaged cross-sectional profile of 9 microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in 2 cells from 1 expanded sample. e , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of the data analyzed in (c) with an average distance of 137.1 ± 10.1 nm (mean ± sd). f , Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g , Zoom in of the white boxed region in (f). h , Average intensity profile of 35 microtubule segments with a length between 1.1 – 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. d , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in (h) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). Scale bars, 2 µm (b,f), 500 nm (c,g).

    Techniques Used: Staining, Labeling, Immunolabeling

    Pre-labeling Ex- d STORM. a , Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b , d STORM image of expanded and re-embedded α− and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c , Zoom in of white boxed region in (b). d , Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5 - 6.4 µm and 28.6 µm in total measured in 4 expanded cells. e , Histogram of peak-to-peak distance distribution of microtubule segments analyzed in (d) with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f , Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g , Magnified view of white boxed region in (f). h , Average cross-sectional profile of 7 microtubule segments with a length between 1.0 – 1.8 µm and 8.7 µm in total. i , Histogram of peak-to-peak distances of the data in (h) with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j , Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k , Zoom-in of white boxed region in (j). l , Average intensity profile of 26 microtubule segments with a length of 2.4 – 10.7 µm and 118.6 µm in total. m , Histogram of peak-to-peak distances determined from microtubule segments in (l) with a mean distance of 226.7 ± 15.3 nm (mean ± sd). n , d STORM image α− and β-tubulin expanded according the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o , Zoom in of white boxed region in (n). p , Average intensity profile of 15 microtubule segments with a length between 1.6-25.1 µm and a total length of 126.0 µm in 1 expanded sample. q , Histogram of peak-to-peak distances determined by fitting the cross-sectional profiles analyzed in (p) with a mean distance of 201.0 ± 9.3 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm (b,f,j,n), 500 nm (c,g,k,o).
    Figure Legend Snippet: Pre-labeling Ex- d STORM. a , Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b , d STORM image of expanded and re-embedded α− and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c , Zoom in of white boxed region in (b). d , Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5 - 6.4 µm and 28.6 µm in total measured in 4 expanded cells. e , Histogram of peak-to-peak distance distribution of microtubule segments analyzed in (d) with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f , Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g , Magnified view of white boxed region in (f). h , Average cross-sectional profile of 7 microtubule segments with a length between 1.0 – 1.8 µm and 8.7 µm in total. i , Histogram of peak-to-peak distances of the data in (h) with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j , Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k , Zoom-in of white boxed region in (j). l , Average intensity profile of 26 microtubule segments with a length of 2.4 – 10.7 µm and 118.6 µm in total. m , Histogram of peak-to-peak distances determined from microtubule segments in (l) with a mean distance of 226.7 ± 15.3 nm (mean ± sd). n , d STORM image α− and β-tubulin expanded according the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o , Zoom in of white boxed region in (n). p , Average intensity profile of 15 microtubule segments with a length between 1.6-25.1 µm and a total length of 126.0 µm in 1 expanded sample. q , Histogram of peak-to-peak distances determined by fitting the cross-sectional profiles analyzed in (p) with a mean distance of 201.0 ± 9.3 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm (b,f,j,n), 500 nm (c,g,k,o).

    Techniques Used: Labeling, Modification, Immunolabeling

    6) Product Images from "The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus"

    Article Title: The BAF (BRG1/BRM-Associated Factor) Chromatin-Remodeling Complex Exhibits Ethanol Sensitivity in Fetal Neural Progenitor Cells and Regulates Transcription at the Mir-9-2 Encoding Gene Locus

    Journal: Alcohol (Fayetteville, N.Y.)

    doi: 10.1016/j.alcohol.2017.01.003

    BRG1 protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).
    Figure Legend Snippet: BRG1 protein expression in NSCs (a) Western immunoblot shows a single BRG1 band at ~238KD detected in the nuclear extract of neurosphere cultures but not in the cytoplasmic extract. (b) BRG1 protein levels in neurospheres after 0mg/dl, 120mg/dl and 320mg/dl ethanol treatment. Lower panel shows the corresponding immunoblot when probed for β-Actin as a loading control. (c) Bar graph, depicting the quantification of the western blot, shows that ethanol did not alter BRG1 protein expression, consistent with the lack of effect on BRG1 mRNA expression. The vertical axis shows the ratio of the band density of BRG1 to the ratio of the band density of β-Actin. Error bars indicates standard error of the mean. (d) Western blot analysis of BRG1 immunoprecipitation in cytoplasmic and nuclear cellular fractions probed with anti-BRG1 antibody. BRG1 is specifically precipitated with anti-BRG1 antibody (blue text), but not with an isotype-specific IgG control antibody (red text). Efficiency of anti-BRG1 immunoprecipitation is indicated by the relative depletion of BRG1 from the nuclear supernatant and enrichment in the immuno-precipitate (IP).

    Techniques Used: Expressing, Western Blot, Immunoprecipitation

    BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.
    Figure Legend Snippet: BRG1-containing complexes associate with both DNAse I-hypersensitive and insensitive sites on the miR-9-2 gene locus (a) Positional representation of POU5F1/Oct4, c-myc and REST transcription regulatory factor binding sites along the human pre-miR-9-2 gene locus identified with the UCSC genome browser ENCODE analysis hub track. Colored circles indicate locations for primer pairs for pri-miR-9-2 regions 1,2,3,4 and 5 and the pre-miR-9-2 coding region. (b) qPCR results of BRG1-ChIP from neurospheres. Primers were used to amplify 6 distinct positions along the pri-miR-9-2 gene locus and a genetically sparse region on mouse chromosome 6. Vertical axis shows fold change relative to an IgG pulldown control. Error bars indicate standard error of the mean. (c) qPCR of neurosphere DNA following digestion with 20, 60 and 120U of DNAse I at the 6 positions along the pri-miR-9-2 gene locus. The vertical axis shows fold amplification relative to DNAse I-untreated neurosphere DNA.

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification

    7) Product Images from "Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons"

    Article Title: Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1628-04.2004

    FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.
    Figure Legend Snippet: FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.

    Techniques Used: Negative Control, Binding Assay, Western Blot, Immunoprecipitation, Molecular Weight, Marker, Immunolabeling, Transfection

    8) Product Images from "Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement"

    Article Title: Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle–mediated release of ezrin to inhibit tumour cell movement

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1692417

    CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p
    Figure Legend Snippet: CD82 and its cholesterol-binding differentially regulate cellular release of EVs. (a) Extracellular staining by filipin and Alexa488-conjugated CTxb in Du145 transfectants. Equal number of the cells were cultured on glass coverslips for 2 days, then fixed and labelled with filipin or Alexa488-conjugated CTxb. For filipin staining, intercellular regions were imaged. For CTxb staining, pericellular regions were imaged. Scale bar: 10 µm. (b) Distributions of Annexin V and Annexin A2 in Du145 transfectant cells. Alexa488-conjugated recombinant Annexin V was used for phosphatidylserine labelling, while Annexin-A2 Ab was used for Annexin-A2 staining. Scale bar: 10 μm. (c) Colocalization of Ezrin with GM1 or Annexin A2 in EVs. For Ezrin and GM1 co-staining, the cells were labelled with the Abs, Alexa488-conjugated CTxB and DAPI. For Ezrin and Annexin A2 co-staining, the cells were incubated sequentially with the primary Abs, Cy3-conjugated donkey anti-goat IgG, normal goat IgG and Alexa594-conjugated goat anti-mouse IgG. Images were obtained by confocal microscopy. Scale bar: 10 µm. (d) The cells were seeded in six-well plate at 50% confluence and cultured in DMEM containing 1% exosome-depleted FBS for 2 – 3 days. The culture supernatants were collected, spun at 2000 × g for 10 min to remove cell debris, and then analysed with NanoSight instrument for EV number and size. Data are presented as mean ± SD (n = 3 individual experiments). *: p

    Techniques Used: Binding Assay, Staining, Cell Culture, Transfection, Recombinant, Incubation, Confocal Microscopy

    9) Product Images from "Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28"

    Article Title: Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

    Journal: mAbs

    doi: 10.4161/mabs.28375

    Figure 6. In vitro response of human PBMC to antagonist anti-CD28 mAbs and ADA . Human PBMC were stimulated with anti-CD3 antibodies and different types of anti-CD28 mAbs plus indicated concentrations of IgG containing ADA. ( A ) Proliferation at
    Figure Legend Snippet: Figure 6. In vitro response of human PBMC to antagonist anti-CD28 mAbs and ADA . Human PBMC were stimulated with anti-CD3 antibodies and different types of anti-CD28 mAbs plus indicated concentrations of IgG containing ADA. ( A ) Proliferation at

    Techniques Used: In Vitro

    10) Product Images from "Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus"

    Article Title: Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

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

    doi: 10.1073/pnas.1513943113

    On the surface of NOD myeloid cells, IgG and Sm show punctate staining. Splenic myeloid cells (CD11b + ) were purified from NOD mice and analyzed for surface levels of Sm and IgG by confocal microscopy; two experiments, two mice, 15–20 cells. Approximately
    Figure Legend Snippet: On the surface of NOD myeloid cells, IgG and Sm show punctate staining. Splenic myeloid cells (CD11b + ) were purified from NOD mice and analyzed for surface levels of Sm and IgG by confocal microscopy; two experiments, two mice, 15–20 cells. Approximately

    Techniques Used: Staining, Purification, Mouse Assay, Confocal Microscopy

    Autoimmune-prone MFs accumulate IgG-ICs on the cell membrane. ( A and B ) CD11b + cells were purified from spleen and analyzed for surface Sm and IgG by confocal imaging. (Scale bars: 2.5 μm.) Data represent two experiments, two mice, 8–10
    Figure Legend Snippet: Autoimmune-prone MFs accumulate IgG-ICs on the cell membrane. ( A and B ) CD11b + cells were purified from spleen and analyzed for surface Sm and IgG by confocal imaging. (Scale bars: 2.5 μm.) Data represent two experiments, two mice, 8–10

    Techniques Used: Purification, Imaging, Mouse Assay

    11) Product Images from "Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman"

    Article Title: Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.162461

    Conceivable functions of cytoplasmic Mbf1 protein via binding to mRNAs. (A) Model for Mbf1-ensured Polycomb silencing. In wild-type and mbf1 mutant lines, Pcm is not upregulated. Therefore, the steady-state level of E(z) mRNA is well balanced irrespective of Mbf1 expression. In Polycomb group mutants, Pcm expression is upregulated so that E(z) mRNA could become susceptible to Pcm attack. However, Mbf1 protects E(z) mRNA to ensure robustness of Polycomb silencing. In mbf1 and Polycomb group double mutants, loss of Mbf1 allows extensive degradation of E(z) mRNA by derepressed Pcm, thereby affecting Polycomb silencing. (B) The enrichment of four representative mRNAs ( GstD5 , Ide , Tep2 and Pebp1 ) identified in the RIP-seq results was confirmed by RIP RT-qPCR analysis. Results for E(z) and RpL30 mRNAs from Fig. 1 F are included for comparison. Data are mean±s.d. of fold-change versus control IgG; * P
    Figure Legend Snippet: Conceivable functions of cytoplasmic Mbf1 protein via binding to mRNAs. (A) Model for Mbf1-ensured Polycomb silencing. In wild-type and mbf1 mutant lines, Pcm is not upregulated. Therefore, the steady-state level of E(z) mRNA is well balanced irrespective of Mbf1 expression. In Polycomb group mutants, Pcm expression is upregulated so that E(z) mRNA could become susceptible to Pcm attack. However, Mbf1 protects E(z) mRNA to ensure robustness of Polycomb silencing. In mbf1 and Polycomb group double mutants, loss of Mbf1 allows extensive degradation of E(z) mRNA by derepressed Pcm, thereby affecting Polycomb silencing. (B) The enrichment of four representative mRNAs ( GstD5 , Ide , Tep2 and Pebp1 ) identified in the RIP-seq results was confirmed by RIP RT-qPCR analysis. Results for E(z) and RpL30 mRNAs from Fig. 1 F are included for comparison. Data are mean±s.d. of fold-change versus control IgG; * P

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Quantitative RT-PCR

    12) Product Images from "The phosphoinositide-binding protein p40phox activates the NADPH oxidase during Fc?IIA receptor-induced phagocytosis"

    Article Title: The phosphoinositide-binding protein p40phox activates the NADPH oxidase during Fc?IIA receptor-induced phagocytosis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20052085

    Expression of FcγIIA receptor, phagocytosis, and NADPH oxidase activity. (A) Analysis of FcγIIA expression by flow cytometry using an FITC-conjugated antibody against CD32 is shown for COS7 and COS phox FcγR cells (left) and human peripheral blood monocytes (right) as indicated. The gray histogram indicates staining of either COS phox FcγR or monocytes with an FITC-conjugated isotype control mAb. (B and C) Phagocytosis of IgG–sheep RBCs in media containing NBT. (B) COS phox FcγR cells incubated with IgG-RBCs for 30 min at 37°C. Many of the ingested IgG-RBCs, which appear tan, are indicated by arrows. (C) Murine bone marrow–derived macrophages incubated with IgG-RBCs for 10 min at 37°C. Formazan-stained phagosomes, indicative of intraphagosomal superoxide production, are indicated by arrows. (D) COS phox FcγR cells incubated with 100 ng/ml phorbol myristate acetate for 30 min, showing diffuse formazan deposits. Bar, 30 μm.
    Figure Legend Snippet: Expression of FcγIIA receptor, phagocytosis, and NADPH oxidase activity. (A) Analysis of FcγIIA expression by flow cytometry using an FITC-conjugated antibody against CD32 is shown for COS7 and COS phox FcγR cells (left) and human peripheral blood monocytes (right) as indicated. The gray histogram indicates staining of either COS phox FcγR or monocytes with an FITC-conjugated isotype control mAb. (B and C) Phagocytosis of IgG–sheep RBCs in media containing NBT. (B) COS phox FcγR cells incubated with IgG-RBCs for 30 min at 37°C. Many of the ingested IgG-RBCs, which appear tan, are indicated by arrows. (C) Murine bone marrow–derived macrophages incubated with IgG-RBCs for 10 min at 37°C. Formazan-stained phagosomes, indicative of intraphagosomal superoxide production, are indicated by arrows. (D) COS phox FcγR cells incubated with 100 ng/ml phorbol myristate acetate for 30 min, showing diffuse formazan deposits. Bar, 30 μm.

    Techniques Used: Expressing, Activity Assay, Flow Cytometry, Cytometry, Staining, Incubation, Derivative Assay

    Expression of p40 phox mutants in COS phox FcγR cells and effect on IgG–sheep RBC–elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblot of cell lysates from COS phox FcγR cells transfected with 0.67 μg of either empty pRK5 or pRK5 containing cDNAs for either wild-type or mutant p40 phox . Blots were probed with antibodies for p40 phox , p47 phox , and p67 phox . (B) COS phox FcγR cells were transfected as in A and incubated with IgG-RBCs in the presence of NBT for 30 min at 37°C. The percentage of cells with NBT + phagosomes is shown as the mean ± SD ( n = 4 except for W207R/D289A, where n = 3). (C) COS phox FcγR cells were transfected as in A for expression of YFP-tagged wild-type or mutant derivatives of p40 phox as indicated or a YFP-tagged PX domain of p40 phox and incubated with IgG–sheep RBCs or with IgG latex beads (*) without or with 50 nM wortmannin, followed by confocal microscopy. Individual phagosomes were scored for either the presence (black bars) or absence of YFP-p40 phox or YFP-p40PX translocation. The number of phagosomes scored for each construct is also shown. Data was collected from two to four independent experiments.
    Figure Legend Snippet: Expression of p40 phox mutants in COS phox FcγR cells and effect on IgG–sheep RBC–elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblot of cell lysates from COS phox FcγR cells transfected with 0.67 μg of either empty pRK5 or pRK5 containing cDNAs for either wild-type or mutant p40 phox . Blots were probed with antibodies for p40 phox , p47 phox , and p67 phox . (B) COS phox FcγR cells were transfected as in A and incubated with IgG-RBCs in the presence of NBT for 30 min at 37°C. The percentage of cells with NBT + phagosomes is shown as the mean ± SD ( n = 4 except for W207R/D289A, where n = 3). (C) COS phox FcγR cells were transfected as in A for expression of YFP-tagged wild-type or mutant derivatives of p40 phox as indicated or a YFP-tagged PX domain of p40 phox and incubated with IgG–sheep RBCs or with IgG latex beads (*) without or with 50 nM wortmannin, followed by confocal microscopy. Individual phagosomes were scored for either the presence (black bars) or absence of YFP-p40 phox or YFP-p40PX translocation. The number of phagosomes scored for each construct is also shown. Data was collected from two to four independent experiments.

    Techniques Used: Expressing, Activity Assay, Transfection, Mutagenesis, Incubation, Confocal Microscopy, Translocation Assay, Construct

    Localization of EYFP-p40PX and full-length EYFP-p40 phox in PLB-985 granulocytes and COS phox FcγR cells during phagocytosis. (A, C, E, G, I, and K) EYFP fluorescence (green). (B, D, F, H, J, and L) EYFP and Alexa Fluor 555 (IgG-RBCs or IgG-beads, red). Regions in which red and green labels overlap appear orange or yellow. (A–D) PLB-985 granulocytes. (E–L) COS phox FcγR cells. (E and F) EYFP-p40PX, IgG-RBCs. (G and H) Full-length EYFP-p40 phox , IgG-RBCs. (A–D and I–L) Full-length EYFP-p40 phox IgG beads without (A, B, I, and J) and with (C, D, K, and L) wortmannin. Images are representative of two to three independent experiments. Arrowheads point to representative phagosomes. Bars: A, 5 μm; C and L, 20 μm. Insets are magnified twofold relative to the adjacent panels.
    Figure Legend Snippet: Localization of EYFP-p40PX and full-length EYFP-p40 phox in PLB-985 granulocytes and COS phox FcγR cells during phagocytosis. (A, C, E, G, I, and K) EYFP fluorescence (green). (B, D, F, H, J, and L) EYFP and Alexa Fluor 555 (IgG-RBCs or IgG-beads, red). Regions in which red and green labels overlap appear orange or yellow. (A–D) PLB-985 granulocytes. (E–L) COS phox FcγR cells. (E and F) EYFP-p40PX, IgG-RBCs. (G and H) Full-length EYFP-p40 phox , IgG-RBCs. (A–D and I–L) Full-length EYFP-p40 phox IgG beads without (A, B, I, and J) and with (C, D, K, and L) wortmannin. Images are representative of two to three independent experiments. Arrowheads point to representative phagosomes. Bars: A, 5 μm; C and L, 20 μm. Insets are magnified twofold relative to the adjacent panels.

    Techniques Used: Fluorescence

    13) Product Images from "m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small cell lung cancer"

    Article Title: m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small cell lung cancer

    Journal: bioRxiv

    doi: 10.1101/2021.07.06.451216

    ALKBH5 altered m 6 A abundance in the 3 untranslated regions of′ specific genes and protein expression ( a ) m 6 A level in the 3 UTRs of target mRNA in PC9 cells transfected with ′siALKBH5#1 or siALKBH5#3 was quantified via MeRIP qPCR using anti-m6A antibody and was compared with that in cell transfected with siNC. The m 6 A level was normalized to that of input fraction (n = 3). IgG was used to evaluate the non-specific binding of the target mRNA. ( b ) A schematic outline showing the workflow for the analysis of downstream targets of ALKBH5. ( c ) Target protein levels in PC9 or A549 cells transfected with siALKBH5#1 or siALKBH5#3 were compared with those transfected with siNC via western blot analysis. Results are presented as mean ± SD. * P
    Figure Legend Snippet: ALKBH5 altered m 6 A abundance in the 3 untranslated regions of′ specific genes and protein expression ( a ) m 6 A level in the 3 UTRs of target mRNA in PC9 cells transfected with ′siALKBH5#1 or siALKBH5#3 was quantified via MeRIP qPCR using anti-m6A antibody and was compared with that in cell transfected with siNC. The m 6 A level was normalized to that of input fraction (n = 3). IgG was used to evaluate the non-specific binding of the target mRNA. ( b ) A schematic outline showing the workflow for the analysis of downstream targets of ALKBH5. ( c ) Target protein levels in PC9 or A549 cells transfected with siALKBH5#1 or siALKBH5#3 were compared with those transfected with siNC via western blot analysis. Results are presented as mean ± SD. * P

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

    14) Product Images from "The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species"

    Article Title: The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.5b00362

    Expression of CvGal2 in Eastern oyster tissues (A) mRNA expression of CvGal2 in different tissues of the eastern oyster was assessed by RT-PCR. Relative expression levels relative to actin are shown. (B) CvGal1 and CvGal2 protein in unattached hemocytes, attached hemocytes, or plasma was detected by Western blot. Recombinant CvGal1 (rCvGal1) and CvGal2 (rCvGal2) were included as specificity controls for the antibody, and a membrane exposed to pre-immune IgG is included as a control for IgG specificity. (C) Hemocytes were allowed to attach for 1 hour, and the presence of CvGal2 on cell surface (w/o Triton) or total (with Triton) was revealed by fluorescence microscopy after antibody staining. Images from cells stained with pre-immune IgG (IgG) are included as controls for IgG specificity. Scale bar: 20μm.
    Figure Legend Snippet: Expression of CvGal2 in Eastern oyster tissues (A) mRNA expression of CvGal2 in different tissues of the eastern oyster was assessed by RT-PCR. Relative expression levels relative to actin are shown. (B) CvGal1 and CvGal2 protein in unattached hemocytes, attached hemocytes, or plasma was detected by Western blot. Recombinant CvGal1 (rCvGal1) and CvGal2 (rCvGal2) were included as specificity controls for the antibody, and a membrane exposed to pre-immune IgG is included as a control for IgG specificity. (C) Hemocytes were allowed to attach for 1 hour, and the presence of CvGal2 on cell surface (w/o Triton) or total (with Triton) was revealed by fluorescence microscopy after antibody staining. Images from cells stained with pre-immune IgG (IgG) are included as controls for IgG specificity. Scale bar: 20μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant, Fluorescence, Microscopy, Staining

    15) Product Images from "Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes"

    Article Title: Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes

    Journal: Growth factors (Chur, Switzerland)

    doi: 10.3109/08977190903407365

    Intestinal histology and morphometric analysis of HB-EGF TG and WT mice. (A) Intestinal histology. Shown are representative H E-stained sections of duodenum from WT, high expression TG mice, and low expression TG mice at 1 month and 5 months of age. The scale bar represents 100 μm. (B) Intestinal morphometric analysis. Villous length, villous width, crypt depth, and muscle thickness in H E-stained sections of duodenum, jejunum, and ileum from WT, high expression TG mice, and low expression TG mice were quantified using ImageJ 1.39U software. (C) Enterocyte immunostaining. Shown are jejunal sections from WT, high expression TG, and low expression TG mice immunostained with rabbit anti-E-cadherin and secondary goat anti-rabbit IgG conjugated with Alexa 560 (red). The nulei were conterstained with DAPI (blue). The scale bar represents 50 μm. (D) Enterocyte cell density. Columnar enterocyte cell densities were quantified by counting the number of cells in the distal 200 μm from the distal tips of the villi. Each graph bar represents quantification of 15 villous tips. All values shown represent mean ± SD. Statistical significance was determined by one-way ANOVA (repeated measures). * p
    Figure Legend Snippet: Intestinal histology and morphometric analysis of HB-EGF TG and WT mice. (A) Intestinal histology. Shown are representative H E-stained sections of duodenum from WT, high expression TG mice, and low expression TG mice at 1 month and 5 months of age. The scale bar represents 100 μm. (B) Intestinal morphometric analysis. Villous length, villous width, crypt depth, and muscle thickness in H E-stained sections of duodenum, jejunum, and ileum from WT, high expression TG mice, and low expression TG mice were quantified using ImageJ 1.39U software. (C) Enterocyte immunostaining. Shown are jejunal sections from WT, high expression TG, and low expression TG mice immunostained with rabbit anti-E-cadherin and secondary goat anti-rabbit IgG conjugated with Alexa 560 (red). The nulei were conterstained with DAPI (blue). The scale bar represents 50 μm. (D) Enterocyte cell density. Columnar enterocyte cell densities were quantified by counting the number of cells in the distal 200 μm from the distal tips of the villi. Each graph bar represents quantification of 15 villous tips. All values shown represent mean ± SD. Statistical significance was determined by one-way ANOVA (repeated measures). * p

    Techniques Used: Mouse Assay, Staining, Expressing, Software, Immunostaining

    16) Product Images from "Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli"

    Article Title: Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli

    Journal: Microorganisms

    doi: 10.3390/microorganisms8081184

    Determination of the presence of EF-Tu, l -asparaginase II and OmpT in outer membrane protein (OMP) extracts and culture supernatants. Protein extracts were separated by SDS-PAGE (12% acrylamide) and analyzed by Western blot assay. Arrows indicate the detection of the corresponding protein. ( A ) Identification of EF-Tu in OMPs and culture supernatant of the EDL933 strain. Monoclonal anti-EF-Tu antibody (mAb 900) was used in a dilution of 1:2000, followed by anti-mouse IgG, HRP conjugate diluted 1:5000. ( B ) Identification of l -asparaginase II in the OMP extracts and culture supernatant obtained from the EDL933, the isogenic mutant ELD933∆ ansB and the complemented ELD933∆ ansB /pVB1_ ansB strains. Anti- l -asparaginase II antibody was used in a dilution of 1:2000, followed by anti-rabbit IgG, HRP conjugate diluted 1:5000. ( C ) Identification of OmpT in the OMP extracts and culture supernatant obtained from the EDL933, the isogenic mutant ELD933∆ ompT and the complemented ELD933∆ ompT /pVB1_ ompT strains. Anti-OmpT antibody was used in a dilution of 1:3000, followed by anti-rabbit IgG, HRP conjugate diluted 1:5000.
    Figure Legend Snippet: Determination of the presence of EF-Tu, l -asparaginase II and OmpT in outer membrane protein (OMP) extracts and culture supernatants. Protein extracts were separated by SDS-PAGE (12% acrylamide) and analyzed by Western blot assay. Arrows indicate the detection of the corresponding protein. ( A ) Identification of EF-Tu in OMPs and culture supernatant of the EDL933 strain. Monoclonal anti-EF-Tu antibody (mAb 900) was used in a dilution of 1:2000, followed by anti-mouse IgG, HRP conjugate diluted 1:5000. ( B ) Identification of l -asparaginase II in the OMP extracts and culture supernatant obtained from the EDL933, the isogenic mutant ELD933∆ ansB and the complemented ELD933∆ ansB /pVB1_ ansB strains. Anti- l -asparaginase II antibody was used in a dilution of 1:2000, followed by anti-rabbit IgG, HRP conjugate diluted 1:5000. ( C ) Identification of OmpT in the OMP extracts and culture supernatant obtained from the EDL933, the isogenic mutant ELD933∆ ompT and the complemented ELD933∆ ompT /pVB1_ ompT strains. Anti-OmpT antibody was used in a dilution of 1:3000, followed by anti-rabbit IgG, HRP conjugate diluted 1:5000.

    Techniques Used: SDS Page, Western Blot, Mutagenesis

    17) Product Images from "Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles"

    Article Title: Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2017.00253

    Expression and surface localization of EGFRvIII epitope in BL21 ΔompA (pET-Nm-fHbpvIII) strain and in its derived outer membrane vesicles (OMVs) . (A) Schematic representation of pET-Nm-fHbpvIII plasmid encoding three copies of EGFRvIIIpep fused to the C-terminus of Neisseria meningitidis fHbp. (B) SDS-PAGE and Western Blot analyses of OMVs. OMVs were purified from BL21 ΔompA (pET21b+) (“Empty” OMVs) and BL21 ΔompA (pET-Nm-fHbpvIII) strains and loaded on SDS-polyacrylamide gels for SDS-PAGE analysis (20 µg OMVs) and Western Blot analysis (1 µg OMVs). After proteins transfer to the nitrocellulose membrane, Nm-fHbp-vIII fusion was visualized using rabbit anti-EGFRvIIIpep antibodies and peroxidase-conjugated anti-rabbit immunoglobulins. (C) Flow cytometry analysis of BL21 ΔompA (pET21b+) and BL21 ΔompA (pET-Nm-fHbpvIII) strains. Bacterial cells were incubated first with anti-EGFRvIIIIpep rabbit antibodies and subsequently with FITC-labeled anti-rabbit secondary antibodies. Fluorescence was measured by flow cytometry. Gray areas represent the background fluorescence signals obtained incubating the cells with the secondary antibody only. (D) Confocal microscopy analysis of BL21 ΔompA (pET21b+) (“Empty” OMVs) and BL21 ΔompA (pET-Nm-fHbpvIII) strains. After induction of protein expression with IPTG, bacterial cells were fixed in 4% formaldehyde solution and incubated first with rabbit anti-EGFRvIIIpep polyclonal antibodies and mouse anti-LPS mAb, and subsequently with goat anti-rabbit IgG, Alexa Fluor 594 conjugated-antibodies (red), and goat anti-mouse IgG, Alexa Fluor 488 conjugated-antibodies (green). (E) Immuno Transmission Electron Microscopy (TEM) analysis of OMVs purified from BL21 ΔompA (pET-Nm-fHbpvIII) strain using primary anti-EGFRvIIIpep rabbit antibodies and 5-nm gold-labeled anti-rabbit secondary antibody (see Materials and Methods for details).
    Figure Legend Snippet: Expression and surface localization of EGFRvIII epitope in BL21 ΔompA (pET-Nm-fHbpvIII) strain and in its derived outer membrane vesicles (OMVs) . (A) Schematic representation of pET-Nm-fHbpvIII plasmid encoding three copies of EGFRvIIIpep fused to the C-terminus of Neisseria meningitidis fHbp. (B) SDS-PAGE and Western Blot analyses of OMVs. OMVs were purified from BL21 ΔompA (pET21b+) (“Empty” OMVs) and BL21 ΔompA (pET-Nm-fHbpvIII) strains and loaded on SDS-polyacrylamide gels for SDS-PAGE analysis (20 µg OMVs) and Western Blot analysis (1 µg OMVs). After proteins transfer to the nitrocellulose membrane, Nm-fHbp-vIII fusion was visualized using rabbit anti-EGFRvIIIpep antibodies and peroxidase-conjugated anti-rabbit immunoglobulins. (C) Flow cytometry analysis of BL21 ΔompA (pET21b+) and BL21 ΔompA (pET-Nm-fHbpvIII) strains. Bacterial cells were incubated first with anti-EGFRvIIIIpep rabbit antibodies and subsequently with FITC-labeled anti-rabbit secondary antibodies. Fluorescence was measured by flow cytometry. Gray areas represent the background fluorescence signals obtained incubating the cells with the secondary antibody only. (D) Confocal microscopy analysis of BL21 ΔompA (pET21b+) (“Empty” OMVs) and BL21 ΔompA (pET-Nm-fHbpvIII) strains. After induction of protein expression with IPTG, bacterial cells were fixed in 4% formaldehyde solution and incubated first with rabbit anti-EGFRvIIIpep polyclonal antibodies and mouse anti-LPS mAb, and subsequently with goat anti-rabbit IgG, Alexa Fluor 594 conjugated-antibodies (red), and goat anti-mouse IgG, Alexa Fluor 488 conjugated-antibodies (green). (E) Immuno Transmission Electron Microscopy (TEM) analysis of OMVs purified from BL21 ΔompA (pET-Nm-fHbpvIII) strain using primary anti-EGFRvIIIpep rabbit antibodies and 5-nm gold-labeled anti-rabbit secondary antibody (see Materials and Methods for details).

    Techniques Used: Expressing, Positron Emission Tomography, Derivative Assay, Plasmid Preparation, SDS Page, Western Blot, Purification, Flow Cytometry, Cytometry, Incubation, Labeling, Fluorescence, Confocal Microscopy, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    18) Product Images from "African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase"

    Article Title: African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

    Journal: Journal of Virology

    doi: 10.1128/JVI.80.7.3157-3166.2006

    Expression and localization of protein pB119L in ASFV-infected Vero cells. (A) Specificity of the anti-GST-B119L antibody in Western blot analysis after reducing SDS-PAGE. Immunoblotting was carried out for mock-infected cells (M) or ASFV-infected cells (I) harvested at 16 hpi. The results with anti-GST-B119L (αB119L) and preimmune serum (PI-B119L) are shown. Molecular mass markers are indicated on the left. (B) Expression of protein pB119L during the ASFV infection. Extracts from mock-infected (Mock) or ASFV-infected cells collected at different hours postinfection were analyzed by Western blotting with the anti-GST-B119L antibody after reducing SDS-PAGE. Results obtained with cells infected for 16 h in the presence of 40 μg of cytosine arabinoside (AraC)/ml are also shown. The band corresponding to protein pB119L is indicated by an arrow. (C) Immunofluorescence microscopy analysis of ASFV-infected Vero cells. Mock-infected (Mock) or ASFV-infected Vero cells (14 hpi) were fixed at 14 hpi and double labeled with anti-GST-B119L antibody detected with Alexa 594 goat anti-rabbit IgG (B119L) and with anti-p72 antibody detected with Alexa 488 goat anti-mouse IgG (p72). Cells were counterstained with DAPI to visualize cellular and viral DNA. Arrows indicate the position of the viral factories.
    Figure Legend Snippet: Expression and localization of protein pB119L in ASFV-infected Vero cells. (A) Specificity of the anti-GST-B119L antibody in Western blot analysis after reducing SDS-PAGE. Immunoblotting was carried out for mock-infected cells (M) or ASFV-infected cells (I) harvested at 16 hpi. The results with anti-GST-B119L (αB119L) and preimmune serum (PI-B119L) are shown. Molecular mass markers are indicated on the left. (B) Expression of protein pB119L during the ASFV infection. Extracts from mock-infected (Mock) or ASFV-infected cells collected at different hours postinfection were analyzed by Western blotting with the anti-GST-B119L antibody after reducing SDS-PAGE. Results obtained with cells infected for 16 h in the presence of 40 μg of cytosine arabinoside (AraC)/ml are also shown. The band corresponding to protein pB119L is indicated by an arrow. (C) Immunofluorescence microscopy analysis of ASFV-infected Vero cells. Mock-infected (Mock) or ASFV-infected Vero cells (14 hpi) were fixed at 14 hpi and double labeled with anti-GST-B119L antibody detected with Alexa 594 goat anti-rabbit IgG (B119L) and with anti-p72 antibody detected with Alexa 488 goat anti-mouse IgG (p72). Cells were counterstained with DAPI to visualize cellular and viral DNA. Arrows indicate the position of the viral factories.

    Techniques Used: Expressing, Infection, Western Blot, SDS Page, Immunofluorescence, Microscopy, Labeling

    19) Product Images from "UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes"

    Article Title: UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-15-34

    GFP-FYVE-UbΔGG expression inhibits endocytosis of EGFR and transferrin receptor in SK-BR-3 cells. GFP or GFP-FYVE-UbΔGG as indicated was expressed in SK-BR-3 cells. A,D. AF-594 transferrin (A) or anti-EGFR antibodies (D) were bound to cells at 4°C, as detailed in Methods, before fixation. B, E. AF-594 transferrin (B) or anti-EGFR antibodies (E) were allowed to internalize at 37°C as detailed in Methods. A,B,D,E; Cells were processed for fluorescence microscopy as in Figure 1 , except that cells shown in A, B, and D were not permeabilized. Tfn, transferrin. Scale bars; 10 μm. D, E. Anti-EGFR antibodies were detected with AF-594-goat anti-mouse IgG. C, F. AF-594-transferrin (C) or AF-594 goat anti-mouse IgG (F) fluorescence was quantitated as described in Methods. In each panel, MFI of cells with surface-bound probes is shown on the left; MFI of cells containing internalized fluorophores is on the right. Values for GFP-expressing cells are shown with gray bars; values for GFP-FYVE-UbΔGG-expressing cells are shown with black bars. Results shown are the average of 2 separate experiments, +/- SEM. The data were compared using a one-way ANOVA test (***p
    Figure Legend Snippet: GFP-FYVE-UbΔGG expression inhibits endocytosis of EGFR and transferrin receptor in SK-BR-3 cells. GFP or GFP-FYVE-UbΔGG as indicated was expressed in SK-BR-3 cells. A,D. AF-594 transferrin (A) or anti-EGFR antibodies (D) were bound to cells at 4°C, as detailed in Methods, before fixation. B, E. AF-594 transferrin (B) or anti-EGFR antibodies (E) were allowed to internalize at 37°C as detailed in Methods. A,B,D,E; Cells were processed for fluorescence microscopy as in Figure 1 , except that cells shown in A, B, and D were not permeabilized. Tfn, transferrin. Scale bars; 10 μm. D, E. Anti-EGFR antibodies were detected with AF-594-goat anti-mouse IgG. C, F. AF-594-transferrin (C) or AF-594 goat anti-mouse IgG (F) fluorescence was quantitated as described in Methods. In each panel, MFI of cells with surface-bound probes is shown on the left; MFI of cells containing internalized fluorophores is on the right. Values for GFP-expressing cells are shown with gray bars; values for GFP-FYVE-UbΔGG-expressing cells are shown with black bars. Results shown are the average of 2 separate experiments, +/- SEM. The data were compared using a one-way ANOVA test (***p

    Techniques Used: Expressing, Fluorescence, Microscopy

    20) Product Images from "Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons"

    Article Title: Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1628-04.2004

    FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.
    Figure Legend Snippet: FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.

    Techniques Used: Negative Control, Binding Assay, Western Blot, Immunoprecipitation, Molecular Weight, Marker, Immunolabeling, Transfection

    21) Product Images from "Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells"

    Article Title: Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells

    Journal: bioRxiv

    doi: 10.1101/2021.01.18.427109

    EPCs express ARG2 and have high levels of ROS a, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs (CD71 + TER119 + ) and RBCs (CD71 − TER119 + ) of control mice (n=6) and NHA (n=6) and HA-PHZ (n=6). Histograms show the representative fluorescence of CellROX Green – FITC in EPCs from the spleen of NHA mouse. P values calculated with unpaired t-test. b, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs, leukocytes (CD45 + ), T-cells (CD45 + CD3e + ), myeloid cells (CD45 + CD11b + ) (n=18). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. c, Percentages of ARG2 + EPCs in control mice (n=11), anemic mice (NHA, n=5; HA-PHZ, n=11; HA-TER119, n=11), neonatal mice (n=5), and isotype control-IgG-treated mice (control-IgG, n=7). d, Percentages of ARG1 + EPCs based on intracellular staining (n=5). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. e, Percentages of YFP + EPCs in reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=8, neonatal n=5, control-IgG n=4, HA-TER119 n=8). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. f, Mean Fluorescence Intensity (MFI) of YFP – FITC in EPCs of reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=4). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. g,h, Total arginase activity in EPCs lysates ( g, n=8) or in the supernatants from EPCs cultures ( h , n=8). P values calculated with one-way ANOVA with unpaired t-test. i, Percentages of ARG1 + EPCs isolated from the spleens of B6.129S4-Arg1 tm1Lky /J incubated with diluent or PHZ (100 μM for 24h) (n=3). P values calculated with one-way ANOVA with unpaired t-test. Data show means ± SD. Each point in a-i represents data from individual mice. n values are the numbers of mice used to obtain the data. The source data underlying Fig.3a-i are provided as a Source Data file.
    Figure Legend Snippet: EPCs express ARG2 and have high levels of ROS a, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs (CD71 + TER119 + ) and RBCs (CD71 − TER119 + ) of control mice (n=6) and NHA (n=6) and HA-PHZ (n=6). Histograms show the representative fluorescence of CellROX Green – FITC in EPCs from the spleen of NHA mouse. P values calculated with unpaired t-test. b, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs, leukocytes (CD45 + ), T-cells (CD45 + CD3e + ), myeloid cells (CD45 + CD11b + ) (n=18). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. c, Percentages of ARG2 + EPCs in control mice (n=11), anemic mice (NHA, n=5; HA-PHZ, n=11; HA-TER119, n=11), neonatal mice (n=5), and isotype control-IgG-treated mice (control-IgG, n=7). d, Percentages of ARG1 + EPCs based on intracellular staining (n=5). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. e, Percentages of YFP + EPCs in reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=8, neonatal n=5, control-IgG n=4, HA-TER119 n=8). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. f, Mean Fluorescence Intensity (MFI) of YFP – FITC in EPCs of reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=4). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. g,h, Total arginase activity in EPCs lysates ( g, n=8) or in the supernatants from EPCs cultures ( h , n=8). P values calculated with one-way ANOVA with unpaired t-test. i, Percentages of ARG1 + EPCs isolated from the spleens of B6.129S4-Arg1 tm1Lky /J incubated with diluent or PHZ (100 μM for 24h) (n=3). P values calculated with one-way ANOVA with unpaired t-test. Data show means ± SD. Each point in a-i represents data from individual mice. n values are the numbers of mice used to obtain the data. The source data underlying Fig.3a-i are provided as a Source Data file.

    Techniques Used: Fluorescence, Mouse Assay, Staining, Activity Assay, Isolation, Incubation

    22) Product Images from "Cytoskeletal Association Is Important for Differential Targeting of Glucose Transporter Isoforms in Leishmania"

    Article Title: Cytoskeletal Association Is Important for Differential Targeting of Glucose Transporter Isoforms in Leishmania

    Journal: The Journal of Cell Biology

    doi:

    Double-label confocal laser scanning micrographs of Triton X-100–extracted L. enriettii promastigotes stained with anti-P1C and anti– α-tubulin. Cytoskeletons were fixed with methanol, stained with a 1:100 dilution of the anti-P1C antibody and a 1:500 dilution of the anti–α-tubulin antibody, and then with an FITC-conjugated anti–rabbit IgG ( P1C ) and a rhodamine-conjugated anti–mouse IgG ( α-tubulin ) secondary antibody. Cytoskeletons were examined by confocal microscopy using illumination at 488 nm to visualize the Pro-1 glucose transporter–complexed FITC antibody ( P1C ) or at 546 nm to visualize α-tubulin complexed with the rhodamine antibody ( α-tubulin ). Each micrograph represents a single 0.5-μm section through each field.
    Figure Legend Snippet: Double-label confocal laser scanning micrographs of Triton X-100–extracted L. enriettii promastigotes stained with anti-P1C and anti– α-tubulin. Cytoskeletons were fixed with methanol, stained with a 1:100 dilution of the anti-P1C antibody and a 1:500 dilution of the anti–α-tubulin antibody, and then with an FITC-conjugated anti–rabbit IgG ( P1C ) and a rhodamine-conjugated anti–mouse IgG ( α-tubulin ) secondary antibody. Cytoskeletons were examined by confocal microscopy using illumination at 488 nm to visualize the Pro-1 glucose transporter–complexed FITC antibody ( P1C ) or at 546 nm to visualize α-tubulin complexed with the rhodamine antibody ( α-tubulin ). Each micrograph represents a single 0.5-μm section through each field.

    Techniques Used: Staining, Confocal Microscopy

    Double-label confocal laser scanning micrographs of Triton X-100–extracted L. enriettii promastigotes transfected with plasmid encoding epitope-tagged iso-1 ( top ) or epitope-tagged iso-2 ( bottom ) and stained with the rabbit anti-GLUT2 antibody directed against the epitope tag ( GLUT2 ) and the murine anti–α-tubulin antibody ( α-tubulin ). Cytoskeletons were fixed with methanol, stained with 1:500 dilutions of the anti-GLUT2 antibody and anti-α-tubulin antibodies, and then with an FITC-conjugated anti–rabbit IgG ( GLUT2 ) and a rhodamine-conjugated anti–mouse IgG ( α-tubulin ) secondary antibody. Cytoskeletons were examined by confocal microscopy. Each micrograph represents a single 0.5-μm section through each field.
    Figure Legend Snippet: Double-label confocal laser scanning micrographs of Triton X-100–extracted L. enriettii promastigotes transfected with plasmid encoding epitope-tagged iso-1 ( top ) or epitope-tagged iso-2 ( bottom ) and stained with the rabbit anti-GLUT2 antibody directed against the epitope tag ( GLUT2 ) and the murine anti–α-tubulin antibody ( α-tubulin ). Cytoskeletons were fixed with methanol, stained with 1:500 dilutions of the anti-GLUT2 antibody and anti-α-tubulin antibodies, and then with an FITC-conjugated anti–rabbit IgG ( GLUT2 ) and a rhodamine-conjugated anti–mouse IgG ( α-tubulin ) secondary antibody. Cytoskeletons were examined by confocal microscopy. Each micrograph represents a single 0.5-μm section through each field.

    Techniques Used: Transfection, Plasmid Preparation, Staining, Confocal Microscopy

    23) Product Images from "YB-1 regulates tumor growth by promoting MACC1/c-Met pathway in human lung adenocarcinoma"

    Article Title: YB-1 regulates tumor growth by promoting MACC1/c-Met pathway in human lung adenocarcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18262

    YB-1 promotes MACC1 transcription by binding to MACC1 promoter and activates MACC1/c-Met pathway (A) Analysis of the MACC1 promoter indicated two putative YB-1 binding sites where the black boxes indicate sequences. RT-PCR analysis (B) and western blot analysis (C) determined the expression of YB-1 and MACC1 after inhibition of YB-1 in A549 cells. (D) MACC1 promoter (-2020 to +262) activity was analyzed by dual-luciferase reporter assay. The YB-1-silenced A549 cells and their corresponding control cells were co-transfected with plvx control plasmid or plvx-YB1 plasmid and MACC1 promoter (-2020 to +262) or basic reporter along with pRL-TK for 24h. (E) Mutated MACC1 promoter plasmids were generated (pGL3- MACC1 -mutated1; pGL3- MACC1 -mutated2). YB-1 binding sites in these two plasmids were mutated at two base pairs (pGL3- MACC1 -mutated1: -1860 to -1856; pGL3- MACC1 -mutated2: -1468 to -1464). A549 cells were transiently co-transfected with plvx vector plasmid or plvx-YB1 plasmid and the pGL3-basic reporter, the wild type MACC1 promoter reporter (pGL3- MACC1 ) or the mutated MACC1 promoter plasmids (pGL3-MACC1-mutated1; pGL3-MACC1-mutated2) along with pRL-TK for 24h. Transfected cells were harvested for dual-luciferase reporter assay. (F) ChIP assay was performed with YB-1 antibody or non-immune IgG as negative control. Immunoprecipitated DNA was amplified by PCR using primers as indicated. (G) Western blot analysis of YB-1, MACC1 and c-Met expression after inhibition of YB-1 in A549 cell. * P
    Figure Legend Snippet: YB-1 promotes MACC1 transcription by binding to MACC1 promoter and activates MACC1/c-Met pathway (A) Analysis of the MACC1 promoter indicated two putative YB-1 binding sites where the black boxes indicate sequences. RT-PCR analysis (B) and western blot analysis (C) determined the expression of YB-1 and MACC1 after inhibition of YB-1 in A549 cells. (D) MACC1 promoter (-2020 to +262) activity was analyzed by dual-luciferase reporter assay. The YB-1-silenced A549 cells and their corresponding control cells were co-transfected with plvx control plasmid or plvx-YB1 plasmid and MACC1 promoter (-2020 to +262) or basic reporter along with pRL-TK for 24h. (E) Mutated MACC1 promoter plasmids were generated (pGL3- MACC1 -mutated1; pGL3- MACC1 -mutated2). YB-1 binding sites in these two plasmids were mutated at two base pairs (pGL3- MACC1 -mutated1: -1860 to -1856; pGL3- MACC1 -mutated2: -1468 to -1464). A549 cells were transiently co-transfected with plvx vector plasmid or plvx-YB1 plasmid and the pGL3-basic reporter, the wild type MACC1 promoter reporter (pGL3- MACC1 ) or the mutated MACC1 promoter plasmids (pGL3-MACC1-mutated1; pGL3-MACC1-mutated2) along with pRL-TK for 24h. Transfected cells were harvested for dual-luciferase reporter assay. (F) ChIP assay was performed with YB-1 antibody or non-immune IgG as negative control. Immunoprecipitated DNA was amplified by PCR using primers as indicated. (G) Western blot analysis of YB-1, MACC1 and c-Met expression after inhibition of YB-1 in A549 cell. * P

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Inhibition, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Generated, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Polymerase Chain Reaction

    24) Product Images from "DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-Hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC"

    Article Title: DC3, the 21-kDa Subunit of the Outer Dynein Arm-Docking Complex (ODA-DC), Is a Novel EF-Hand Protein Important for Assembly of Both the Outer Arm and the ODA-DC

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-01-0057

    (A) DC1 and DC2 assemble on the axoneme in the absence of DC3, but not vice versa. Top panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, and oda 3 were isolated and analyzed by western blotting. oda 1 is null for DC2; oda 3 is null for DC1. The blot was probed with antibodies to DC1, DC2, and the inner arm IC, IC140 (used as a loading control). As expected, antibodies to DC1 detected protein in wild-type axonemes, but not in oda 1 or oda 3 axonemes, which lack the ODA-DC. Importantly, the antibody also detected protein in axonemes of the DC3-deletion strain. Essentially identical results were obtained with antibodies to DC2 (our unpublished results). These data indicate that DC1 and DC2 can assemble on the axoneme in the complete absence of DC3. Bottom panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, oda 3, and oda 9 were prepared as above ( oda 9 has a defect in IC1 and lacks outer dynein arms but retains the ODA-DC). The blot was probed with a polyclonal antibody to DC3. The antibody detects a single protein of M r ∼25,000 in both oda 9 and wild-type axonemes but detects no protein in DC3-null axonemes, indicating that it is specific for DC3. Axonemes from oda 1 and oda 3 do not contain DC3, indicating that DC3 assembly is dependent on the presence of DC1 and DC2. (B) Immunoprecipitation of the ODA-DC in the absence of Mg 2 + . The DC1 antibody was used to immunoprecipitate the ODA-DC from biotinylated 0.6 M KCl extracts of DC3-transformant (strain W215) and DC3-null axonemes. The immunoprecipitated proteins were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using streptavidin-HRP. The anti-DC1 antibody immuno-precipitated three proteins of M r ∼105,000, ∼70,000, and ∼25,000 (arrowheads) from DC3-transformant axonemal extracts (lane 3). These proteins, corresponding to DC1, DC2, and DC3, respectively, were not immunoprecipitated from DC3-transformant axonemal extracts using normal rabbit IgG (lane 4). In contrast, the anti-DC1 antibody immunoprecipitated only DC1 and DC2 (arrowheads) from the DC3-null axonemal extracts (lane 1). These proteins were not immunoprecipitated from DC3-null axonemal extracts using normal rabbit IgG (lane 2). These data confirm that a “partial” docking complex composed of DC1 and DC2 assembles on the axoneme when DC3 is missing and that transformation of the DC3-deletion strain with the DC3 gene restores DC3 to the ODA-DC. Numbers on left indicate molecular weight markers.
    Figure Legend Snippet: (A) DC1 and DC2 assemble on the axoneme in the absence of DC3, but not vice versa. Top panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, and oda 3 were isolated and analyzed by western blotting. oda 1 is null for DC2; oda 3 is null for DC1. The blot was probed with antibodies to DC1, DC2, and the inner arm IC, IC140 (used as a loading control). As expected, antibodies to DC1 detected protein in wild-type axonemes, but not in oda 1 or oda 3 axonemes, which lack the ODA-DC. Importantly, the antibody also detected protein in axonemes of the DC3-deletion strain. Essentially identical results were obtained with antibodies to DC2 (our unpublished results). These data indicate that DC1 and DC2 can assemble on the axoneme in the complete absence of DC3. Bottom panel: Axonemes from wild type, the DC3-deletion strain (DC3Δ), oda 1, oda 3, and oda 9 were prepared as above ( oda 9 has a defect in IC1 and lacks outer dynein arms but retains the ODA-DC). The blot was probed with a polyclonal antibody to DC3. The antibody detects a single protein of M r ∼25,000 in both oda 9 and wild-type axonemes but detects no protein in DC3-null axonemes, indicating that it is specific for DC3. Axonemes from oda 1 and oda 3 do not contain DC3, indicating that DC3 assembly is dependent on the presence of DC1 and DC2. (B) Immunoprecipitation of the ODA-DC in the absence of Mg 2 + . The DC1 antibody was used to immunoprecipitate the ODA-DC from biotinylated 0.6 M KCl extracts of DC3-transformant (strain W215) and DC3-null axonemes. The immunoprecipitated proteins were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using streptavidin-HRP. The anti-DC1 antibody immuno-precipitated three proteins of M r ∼105,000, ∼70,000, and ∼25,000 (arrowheads) from DC3-transformant axonemal extracts (lane 3). These proteins, corresponding to DC1, DC2, and DC3, respectively, were not immunoprecipitated from DC3-transformant axonemal extracts using normal rabbit IgG (lane 4). In contrast, the anti-DC1 antibody immunoprecipitated only DC1 and DC2 (arrowheads) from the DC3-null axonemal extracts (lane 1). These proteins were not immunoprecipitated from DC3-null axonemal extracts using normal rabbit IgG (lane 2). These data confirm that a “partial” docking complex composed of DC1 and DC2 assembles on the axoneme when DC3 is missing and that transformation of the DC3-deletion strain with the DC3 gene restores DC3 to the ODA-DC. Numbers on left indicate molecular weight markers.

    Techniques Used: Isolation, Western Blot, Immunoprecipitation, SDS Page, Transformation Assay, Molecular Weight

    25) Product Images from "FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis"

    Article Title: FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2017.00576

    Co-immunoprecipitation (Co-IP) of TnFXYD8 and NKA α-subunit (NKA α) in the pufferfish kidneys. TnFXYD8 or NKA were immunoprecipitated from renal crude membrane fractions of freshwater pufferfish using primary antibodies, then the immune complexes were analyzed by immunoblotting for NKA α (A) or TnFXYD8 (B) , respectively. In immunoprecipitated NKA α or TnFXYD8, the immunoblotting analyses for NKAα and TnFXYD8 revealed immunoreactive bands at 100 kDa (A) and 13 kDa (B) , respectively. In ( B) , the 55-kDa band in lane 3 is the IgG heavy chain of TnFXYD8 antibody. M, marker (kDa); lane 1, immunoblot detection of the opposing antibody (experimental group); lane 2, negative control for no antibody incubation in IP; lane 3, positive control using the same antibody with IP.
    Figure Legend Snippet: Co-immunoprecipitation (Co-IP) of TnFXYD8 and NKA α-subunit (NKA α) in the pufferfish kidneys. TnFXYD8 or NKA were immunoprecipitated from renal crude membrane fractions of freshwater pufferfish using primary antibodies, then the immune complexes were analyzed by immunoblotting for NKA α (A) or TnFXYD8 (B) , respectively. In immunoprecipitated NKA α or TnFXYD8, the immunoblotting analyses for NKAα and TnFXYD8 revealed immunoreactive bands at 100 kDa (A) and 13 kDa (B) , respectively. In ( B) , the 55-kDa band in lane 3 is the IgG heavy chain of TnFXYD8 antibody. M, marker (kDa); lane 1, immunoblot detection of the opposing antibody (experimental group); lane 2, negative control for no antibody incubation in IP; lane 3, positive control using the same antibody with IP.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Marker, Negative Control, Incubation, Positive Control

    26) Product Images from "Ubiquitin-Specific Protease 25 Regulates TLR4-Dependent Innate Immune Responses Through Deubiquitination of the Adaptor Protein TRAF3"

    Article Title: Ubiquitin-Specific Protease 25 Regulates TLR4-Dependent Innate Immune Responses Through Deubiquitination of the Adaptor Protein TRAF3

    Journal: Science signaling

    doi: 10.1126/scisignal.2003708

    USP25 cleaves the cIAP2-mediated K 48 -linked ubiquitin chain from TRAF3 ( A and B ) USP25 cleaves K 48 -linked ubiquitin chains from TRAF3. HEK 293T cells were transfected with plasmids encoding the indicated constructs. Twenty hours later, cells were lysed and the cell lysates were denatured in 1% SDS and then heated to 95°C for 5 min. The denatured lysates were subjected to immunoprecipitation (denature-IP) with the control IgG (Ig) or an anti-FLAG antibody (αF). The immunoprecipitates were analyzed by Western blotting with antibodies against (A) ubiquitin (αUbi, top blot), (B) hemagglutinin (HA; αHA, top blot), or (A and B) FLAG (middle blots). The abundances of the indicated proteins in the cell lysates were determined by Western blotting analysis with an anti-Myc antibody (αMyc, bottom blots). ( C ) USP25 specifically cleaves K 63 -linked ubiquitin chains from TRAF3 in vitro. The strategy to obtain TRAF3 protein that was modified with either K 63 - or K 48 -linked ubiquitin chains and then combine them with USP25 or USP25(C178S) proteins is illustrated (also see Materials and Methods for further details). A deubiquitination assay was performed by mixing the indicated proteins followed by Western blotting analysis with antibodies against HA, FLAG, or USP25. exp., exposure. ( D ) Deficiency in USP25 results in increased K 48 -linked ubiquitination of TRAF3 after stimulation with LPS. WT and Usp25 −/− BMDMs were treated with LPS (10 μg/ml) for the indicated times. The cells were lysed, and the cell lysates were subjected to denaturing IP with an anti-TRAF3 antibody. The immunoprecipitated samples were subjected to Western blotting analysis with antibodies specific for K 48 - or K 63 -linked ubiquitin (top two blots). The abundances of the indicated proteins in the cell lysates were determined by Western blotting analysis with specific antibodies against the indicated proteins (bottom blot). Data are representative of three (A and B) or two (C and D) independent experiments.
    Figure Legend Snippet: USP25 cleaves the cIAP2-mediated K 48 -linked ubiquitin chain from TRAF3 ( A and B ) USP25 cleaves K 48 -linked ubiquitin chains from TRAF3. HEK 293T cells were transfected with plasmids encoding the indicated constructs. Twenty hours later, cells were lysed and the cell lysates were denatured in 1% SDS and then heated to 95°C for 5 min. The denatured lysates were subjected to immunoprecipitation (denature-IP) with the control IgG (Ig) or an anti-FLAG antibody (αF). The immunoprecipitates were analyzed by Western blotting with antibodies against (A) ubiquitin (αUbi, top blot), (B) hemagglutinin (HA; αHA, top blot), or (A and B) FLAG (middle blots). The abundances of the indicated proteins in the cell lysates were determined by Western blotting analysis with an anti-Myc antibody (αMyc, bottom blots). ( C ) USP25 specifically cleaves K 63 -linked ubiquitin chains from TRAF3 in vitro. The strategy to obtain TRAF3 protein that was modified with either K 63 - or K 48 -linked ubiquitin chains and then combine them with USP25 or USP25(C178S) proteins is illustrated (also see Materials and Methods for further details). A deubiquitination assay was performed by mixing the indicated proteins followed by Western blotting analysis with antibodies against HA, FLAG, or USP25. exp., exposure. ( D ) Deficiency in USP25 results in increased K 48 -linked ubiquitination of TRAF3 after stimulation with LPS. WT and Usp25 −/− BMDMs were treated with LPS (10 μg/ml) for the indicated times. The cells were lysed, and the cell lysates were subjected to denaturing IP with an anti-TRAF3 antibody. The immunoprecipitated samples were subjected to Western blotting analysis with antibodies specific for K 48 - or K 63 -linked ubiquitin (top two blots). The abundances of the indicated proteins in the cell lysates were determined by Western blotting analysis with specific antibodies against the indicated proteins (bottom blot). Data are representative of three (A and B) or two (C and D) independent experiments.

    Techniques Used: Transfection, Construct, Immunoprecipitation, Western Blot, In Vitro, Modification

    27) Product Images from "Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA"

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

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiy467

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

    Techniques Used: Binding Assay, Infection, Inhibition, Incubation

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

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

    28) Product Images from "Control of somatic tissue differentiation by the long non-coding RNA TINCR"

    Article Title: Control of somatic tissue differentiation by the long non-coding RNA TINCR

    Journal: Nature

    doi: 10.1038/nature11661

    TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.
    Figure Legend Snippet: TINCR interacts with differentiation mRNAs and STAU1 protein a , Enriched GO terms in TINCR-interacting genes detected by RIA-Seq. b , Protein microarray analysis detects TINCR RNA binding to STAU1 protein. Human recombinant protein microarray spotted with approximately 9,400 proteins (left); enlarged 144 protein spot subarray (middle) demonstrating strand-specific binding of TINCR sense strand to STAU1 protein (right); DUPD1 protein negative control is shown. Alexa-Fluor-647-labelled rabbit anti-mouse IgG in the top left corner of each subarray. c , STAU1 protein immunoprecipitation pulls down TINCR RNA. ANCR and LINC1 (also known as XIST ) represent lncRNA controls. d , Streptavidin precipitation of in vitro synthesized biotinylated TINCR RNA pulls down STAU1 protein. HA, haemagglutinin; WB, western blot.

    Techniques Used: Microarray, RNA Binding Assay, Recombinant, Binding Assay, Negative Control, Immunoprecipitation, In Vitro, Synthesized, Western Blot

    29) Product Images from "Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons"

    Article Title: Fibroblast Growth Factor Homologous Factor 2B: Association with Nav1.6 and Selective Colocalization at Nodes of Ranvier of Dorsal Root Axons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1628-04.2004

    FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.
    Figure Legend Snippet: FHF2 and Na v 1.6 are colocalized in the brain. A , Pan sodium channel and FHF2 antibodies were used to immunoprecipitate (IP) the voltage-gated sodium channels from a rat brain lysate. Anti-mouse IgG was used as a negative control to rule out nonspecific binding. Western blotting analysis of the IP complex was performed by the pan sodium channel antibody. Lane 1 shows a robust immunoreactive signal from the cell lysate that was used for the IP assay, consistent with the presence of intact sodium channel proteins in this sample. Nonspecific antibodies do not immunoprecipitate a channel complex (lane 2). As predicted, pan sodium channel antibody immunoprecipitated a channel complex (lane 3). The reduced signal in lane 3 compared with lane 1 is likely attributable to the avidity of the antibody in the two different assays. FHF2 coimmunoprecipitated voltage-gated sodium channels from the brain lysate (lane 4). Comparison of the immunoreactive bands in lanes 1 and 4 might be explained by the interaction of FHF2 with only a subset of the cellular pool of channels. The molecular weight marker (in kilodaltons) is shown on the left. B , Colocalization of Na v 1.6 and FHF2 in the hippocampus. Na v 1.6- and FHF2-specific antibodies were used to immunolabel sections of rat hippocampus. FHF2 (green) and Na v 1.6 (red) proteins were detected in the pyramidal cells of Ammon's horn, and the merged images (yellow) show significant colocalization of the two proteins. The inset shows the pyramidal cells at 40× magnification. C , GFP antibody was used to IP Na v 1.6 from lysates of HEK 293 cells transfected with either Na v 1.6 plus GFP (control; lane 3) or Na v 1.6 plus FHF2B-GFP (lane 4). The IP complex was probed with the pan sodium channel antibody and detected no association between Na v 1.6 with GFP (lane 3), but an association of Na v 1.6 with FHF2B (lane 4). Lanes 1 and 2 show Western blotting analysis of the cell lysates probed with pan sodium channel antibody (top) and GFP (bottom) to show comparable levels of Na v 1.6/GFP (lane 1) and Na v 1.6/FHF2B-GFP (lane 2) in the samples used for the immunoprecipitation assay.

    Techniques Used: Negative Control, Binding Assay, Western Blot, Immunoprecipitation, Molecular Weight, Marker, Immunolabeling, Transfection

    30) Product Images from "Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A"

    Article Title: Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A

    Journal: Journal of Virology

    doi: 10.1128/JVI.05771-11

    Comparison of the Tva display on the surface of CEFs from CB, L15, and P chicken lines. Cells were incubated with crude extracellular supernatants containing the SU(A)-rIgG immunoadhesin and with goat-anti-rabbit IgG conjugated with Alexa Fluor 488. The
    Figure Legend Snippet: Comparison of the Tva display on the surface of CEFs from CB, L15, and P chicken lines. Cells were incubated with crude extracellular supernatants containing the SU(A)-rIgG immunoadhesin and with goat-anti-rabbit IgG conjugated with Alexa Fluor 488. The

    Techniques Used: Incubation

    31) Product Images from "Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG"

    Article Title: Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031588

    Indirect immunofluorescence microscopy (A,B,C D). Anti-Msp1 (A B) and anti-Msp2 (C D) rabbit antisera were used on wild-type (A C) en msp1 mutant cells (B D). Anti-rabbit IgG antibodies conjugated with Alexa Fluor 488 (Invitrogen) were used to visualize the Msp1 and Msp2 localization on the cells.
    Figure Legend Snippet: Indirect immunofluorescence microscopy (A,B,C D). Anti-Msp1 (A B) and anti-Msp2 (C D) rabbit antisera were used on wild-type (A C) en msp1 mutant cells (B D). Anti-rabbit IgG antibodies conjugated with Alexa Fluor 488 (Invitrogen) were used to visualize the Msp1 and Msp2 localization on the cells.

    Techniques Used: Immunofluorescence, Microscopy, Mutagenesis

    32) Product Images from "The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme"

    Article Title: The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001933

    Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.
    Figure Legend Snippet: Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation

    33) Product Images from "Antioxidant defense by thioredoxin can occur independently of canonical thiol-disulfide oxidoreductase enzymatic activity"

    Article Title: Antioxidant defense by thioredoxin can occur independently of canonical thiol-disulfide oxidoreductase enzymatic activity

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.02.066

    SsrB is a substrate of thioredoxin-1 thiol-disulfide oxidoreductase-dependent and –independent activities Western blotting of SsrB-3XFLAG after lysates of Salmonella containing pTRXATAP plasmids were purified sequentially with IgG (1 st elution) and calmodulin (2 nd elution) (A). Detection of the purified TrxA-6His proteins bound to the full-length of GST-SsrB by anti-6His immunoblot analysis after pull-down. The GST protein was used as control (B). Molecular markers are indicated. A recombinant C-terminal domain of SsrB separated by PAGE gel electrophoresis was visualized by Coomassie brilliant blue staining (C). Where indicated, 25 µM of the C-terminal domain of SsrB were oxidized with 250 µM H 2 O 2 . Some of the specimens exposed to H 2 O 2 were treated with 25 µM of recombinant TrxA or TrxA C32A C35A. The sizes of SsrB monomers and dimers are indicated on the right. TrxA-6His and TrxA C32A C35A-6His proteins were visualized by Western blot using an anti-6His antibody after the pull-down with recombinant GST-SsrB (D). Interactions between wild-type or TrxA C32A C35A with full-length SsrB were studied in a bacterial two-hybrid system that reconstitutes the T18 and T25 domains of adenylate cyclase (E). Thioredoxin reductase (TrxB) and the RpoA α-subunit of the RNA polymerase were included as positive and negative controls, respectively. The activity of reconstituted adenylate cyclase is expressed in Miller units (M.U.). Abundance of TrxA in soluble and whole cell cytoplasmic extracts of stationary phase Salmonella was determined by Western blotting (F). The amount of ssrB mRNA was quantified in overnight cultures of WT and Δ trxA Salmonella (G). The data are expressed relative to the rpoD .
    Figure Legend Snippet: SsrB is a substrate of thioredoxin-1 thiol-disulfide oxidoreductase-dependent and –independent activities Western blotting of SsrB-3XFLAG after lysates of Salmonella containing pTRXATAP plasmids were purified sequentially with IgG (1 st elution) and calmodulin (2 nd elution) (A). Detection of the purified TrxA-6His proteins bound to the full-length of GST-SsrB by anti-6His immunoblot analysis after pull-down. The GST protein was used as control (B). Molecular markers are indicated. A recombinant C-terminal domain of SsrB separated by PAGE gel electrophoresis was visualized by Coomassie brilliant blue staining (C). Where indicated, 25 µM of the C-terminal domain of SsrB were oxidized with 250 µM H 2 O 2 . Some of the specimens exposed to H 2 O 2 were treated with 25 µM of recombinant TrxA or TrxA C32A C35A. The sizes of SsrB monomers and dimers are indicated on the right. TrxA-6His and TrxA C32A C35A-6His proteins were visualized by Western blot using an anti-6His antibody after the pull-down with recombinant GST-SsrB (D). Interactions between wild-type or TrxA C32A C35A with full-length SsrB were studied in a bacterial two-hybrid system that reconstitutes the T18 and T25 domains of adenylate cyclase (E). Thioredoxin reductase (TrxB) and the RpoA α-subunit of the RNA polymerase were included as positive and negative controls, respectively. The activity of reconstituted adenylate cyclase is expressed in Miller units (M.U.). Abundance of TrxA in soluble and whole cell cytoplasmic extracts of stationary phase Salmonella was determined by Western blotting (F). The amount of ssrB mRNA was quantified in overnight cultures of WT and Δ trxA Salmonella (G). The data are expressed relative to the rpoD .

    Techniques Used: Western Blot, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Nucleic Acid Electrophoresis, Staining, Activity Assay

    34) Product Images from "Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1"

    Article Title: Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0900983

    A , K562 cells expressing α M -mCFP and β 2 -mYFP were labeled with anti-α M ICRF44 mAb, anti-β 2 TS1/18 mAb, or IgG1 isotype mAb (red trace), followed by PE-conjugated secondary Abs. Cells were analyzed by flow cytometry. B , K562 cells expressing α M -mCFP and β 2 -mYFP were visualized under phase contrast and immunofluorescence microscopy. C , Whole-cell lysates from K562 cells expressing α M -mCFP and β 2 -mYFP were analyzed by immunoblotting using an anti-GFP polyclonal Ab. D , Mac-1 was immunoprecipitated from K562 cells, K562 cells expressing wild-type α M and β 2 , and K562 cells expressing α M -mCFP and β 2 -mYFP using the anti-β 2 CBR LFA-1/2 mAb. Proteins were separated on a 7.5% SDS-PAGE gel and then analyzed by silver staining. E , K562 cells expressing α M -mCFP and β 2 -mYFP were allowed to adhere to tissue culture plastic coated with ICAM-1 or PVP, as a control, in the presence or absence of 1 mM MnCl 2 , 10 µg/ml CBR LFA-1/2 mAb, and anti-α M clone 44 mAb. After a 30-min incubation, nonadherent cells were washed away, and the number of adherent cells per field was counted. Data are presented as mean ± SEM from one of three independent experiments. *, Significantly different from control ( p
    Figure Legend Snippet: A , K562 cells expressing α M -mCFP and β 2 -mYFP were labeled with anti-α M ICRF44 mAb, anti-β 2 TS1/18 mAb, or IgG1 isotype mAb (red trace), followed by PE-conjugated secondary Abs. Cells were analyzed by flow cytometry. B , K562 cells expressing α M -mCFP and β 2 -mYFP were visualized under phase contrast and immunofluorescence microscopy. C , Whole-cell lysates from K562 cells expressing α M -mCFP and β 2 -mYFP were analyzed by immunoblotting using an anti-GFP polyclonal Ab. D , Mac-1 was immunoprecipitated from K562 cells, K562 cells expressing wild-type α M and β 2 , and K562 cells expressing α M -mCFP and β 2 -mYFP using the anti-β 2 CBR LFA-1/2 mAb. Proteins were separated on a 7.5% SDS-PAGE gel and then analyzed by silver staining. E , K562 cells expressing α M -mCFP and β 2 -mYFP were allowed to adhere to tissue culture plastic coated with ICAM-1 or PVP, as a control, in the presence or absence of 1 mM MnCl 2 , 10 µg/ml CBR LFA-1/2 mAb, and anti-α M clone 44 mAb. After a 30-min incubation, nonadherent cells were washed away, and the number of adherent cells per field was counted. Data are presented as mean ± SEM from one of three independent experiments. *, Significantly different from control ( p

    Techniques Used: Expressing, Labeling, Flow Cytometry, Cytometry, Immunofluorescence, Microscopy, Immunoprecipitation, SDS Page, Silver Staining, Incubation

    35) Product Images from "A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification"

    Article Title: A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115046

    The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.
    Figure Legend Snippet: The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.

    Techniques Used: Concentration Assay, Microscopy, Standard Deviation

    Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.
    Figure Legend Snippet: Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Techniques Used: Labeling, Biomarker Assay, Concentration Assay

    Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.
    Figure Legend Snippet: Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Techniques Used: Fluorescence, Microscopy, Labeling, Biomarker Assay, Concentration Assay

    36) Product Images from "Effects of heparin on the uptake of lipoprotein lipase in rat liver"

    Article Title: Effects of heparin on the uptake of lipoprotein lipase in rat liver

    Journal: BMC Physiology

    doi: 10.1186/1472-6793-4-13

    Distribution of bovine LPL in livers at different times after injection, and the effect of heparin . Tissue sections were stained with the rabbit polyclonal anti-LPL IgG and then with goat anti-rabbit IgG labelled with Alexa fluor 488 (green). Panels A, C and E are from rats that did not receive heparin. Panels B, D and F are from rats that had been given heparin five min before the injection of active bovine LPL. Panels A and B are two min, panels C and D are 15 min, and panels E and F are 60 min after injection of the lipase. The inset in panel E shows a section from the liver of a rat that was not injected with bovine LPL. The magnification was × 20.
    Figure Legend Snippet: Distribution of bovine LPL in livers at different times after injection, and the effect of heparin . Tissue sections were stained with the rabbit polyclonal anti-LPL IgG and then with goat anti-rabbit IgG labelled with Alexa fluor 488 (green). Panels A, C and E are from rats that did not receive heparin. Panels B, D and F are from rats that had been given heparin five min before the injection of active bovine LPL. Panels A and B are two min, panels C and D are 15 min, and panels E and F are 60 min after injection of the lipase. The inset in panel E shows a section from the liver of a rat that was not injected with bovine LPL. The magnification was × 20.

    Techniques Used: Injection, Staining

    Detection of endogenous LPL in rat liver by immunofluorescence . Tissue sections were double-stained with chicken anti-LPL IgG (detected with Alexa 488-labeled goat anti-chicken antibodies (green)), and the monoclonal anti-Kupffer cell antibody ED2 (detected with Alexa 546-labeled goat anti-mouse antibodies (red)). All panels show sections from livers of rats that did not receive heparin. The magnification in panels A and B was × 20-fold whereas it was × 60 + zooming in panel C. Panel A shows staining only for LPL. Panel B is the same area as in A but with staining also for ED2. Panel C shows both stainings. The inset in panel A shows a control section with non-immune chicken IgG instead of anti-LPL.
    Figure Legend Snippet: Detection of endogenous LPL in rat liver by immunofluorescence . Tissue sections were double-stained with chicken anti-LPL IgG (detected with Alexa 488-labeled goat anti-chicken antibodies (green)), and the monoclonal anti-Kupffer cell antibody ED2 (detected with Alexa 546-labeled goat anti-mouse antibodies (red)). All panels show sections from livers of rats that did not receive heparin. The magnification in panels A and B was × 20-fold whereas it was × 60 + zooming in panel C. Panel A shows staining only for LPL. Panel B is the same area as in A but with staining also for ED2. Panel C shows both stainings. The inset in panel A shows a control section with non-immune chicken IgG instead of anti-LPL.

    Techniques Used: Immunofluorescence, Staining, Labeling

    37) Product Images from "Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal"

    Article Title: Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003513

    Binding of mouse FH to A. stephensi mosquito proventriculus and anterior midgut epithelial surface in indirect immunofluorescence assays. Female A. stephensi mosquitoes were fed on mouse blood using the membrane glass feeder and were cut thereafter into sections. (A) An overlay image showing binding of mouse FH from the blood meal to the epithelial surface of the proventriculus (oval shaped region) and anterior midgut (narrow tube region) of the mosquito as a green fluorescence and DAPI nuclear staining as a blue fluorescence. (B) An overlay image of a consecutive mosquito section probed with Alexa Fluor 488 Donkey anti-goat IgG only to control for cross reactivity towards mosquito and blood proteins in the absence of anti-FH. Absence of green fluorescence in (B) indicated lack of cross reactivity. Scale bars are 50 μm.
    Figure Legend Snippet: Binding of mouse FH to A. stephensi mosquito proventriculus and anterior midgut epithelial surface in indirect immunofluorescence assays. Female A. stephensi mosquitoes were fed on mouse blood using the membrane glass feeder and were cut thereafter into sections. (A) An overlay image showing binding of mouse FH from the blood meal to the epithelial surface of the proventriculus (oval shaped region) and anterior midgut (narrow tube region) of the mosquito as a green fluorescence and DAPI nuclear staining as a blue fluorescence. (B) An overlay image of a consecutive mosquito section probed with Alexa Fluor 488 Donkey anti-goat IgG only to control for cross reactivity towards mosquito and blood proteins in the absence of anti-FH. Absence of green fluorescence in (B) indicated lack of cross reactivity. Scale bars are 50 μm.

    Techniques Used: Binding Assay, Immunofluorescence, Fluorescence, Staining

    38) Product Images from "Generation and Characterization of a Diabody Targeting the ?v?6 Integrin"

    Article Title: Generation and Characterization of a Diabody Targeting the ?v?6 Integrin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073260

    Treatment of α v β 6 -expressing cells with B6.3 diabody resulted in diabody internalization and blockade of integrin functions. A) Localization of B6.3 diabody in A375Pβ6 cells by confocal microscopy. B6.3 diabody detection showed membrane pattern of staining at 4°C and internalized when cells were incubated at 37°C for 30 min, 1 h and 3 h. B6.3 diabody was detected using rabbit anti-human IgG followed by Alexa Fluor 546®-labeled goat anti-rabbit IgG (red). Cells were also counterstained with Hoechst 33245 (blue). B) Treatment of α v β 6 -expressing cells blocked adhesion to LAP-coated plates (A375Pβ6 and Capan-1 cells) and/or fibronectin-coated plates (Capan-1 cells). Cells were incubated with B6.3 or shMFE23 diabody at 4°C for 1 h and allowed to attach to coated plates for 1 h at 37°C. Treatment with the anti-CEA shMFE23 diabody had no effect on the cell lines used. C) B6.3 diabody treatment inhibited migration towards LAP and fibronectin. As observed in adhesion assays, the diabody inhibited migration of A375Pβ6 cells to LAP and migration of Capan-1 cells to fibronectin and LAP, while targeting CEA had no effect on the cells tested.
    Figure Legend Snippet: Treatment of α v β 6 -expressing cells with B6.3 diabody resulted in diabody internalization and blockade of integrin functions. A) Localization of B6.3 diabody in A375Pβ6 cells by confocal microscopy. B6.3 diabody detection showed membrane pattern of staining at 4°C and internalized when cells were incubated at 37°C for 30 min, 1 h and 3 h. B6.3 diabody was detected using rabbit anti-human IgG followed by Alexa Fluor 546®-labeled goat anti-rabbit IgG (red). Cells were also counterstained with Hoechst 33245 (blue). B) Treatment of α v β 6 -expressing cells blocked adhesion to LAP-coated plates (A375Pβ6 and Capan-1 cells) and/or fibronectin-coated plates (Capan-1 cells). Cells were incubated with B6.3 or shMFE23 diabody at 4°C for 1 h and allowed to attach to coated plates for 1 h at 37°C. Treatment with the anti-CEA shMFE23 diabody had no effect on the cell lines used. C) B6.3 diabody treatment inhibited migration towards LAP and fibronectin. As observed in adhesion assays, the diabody inhibited migration of A375Pβ6 cells to LAP and migration of Capan-1 cells to fibronectin and LAP, while targeting CEA had no effect on the cells tested.

    Techniques Used: Expressing, Confocal Microscopy, Staining, Incubation, Labeling, Migration

    B6.3 diabody inhibited LAP-mediated Smad2/3 translocation to the nucleus in Capan-1 cells. Cells were incubated at 4°C in the presence of B6.3 diabody and then treated with LAP or TGFβ 1 (30 min, 37°C); Smad2/3 localization was assessed by confocal microscopy (40X) using rabbit anti-Smad2/3 followed by Alexa Fluor 488®-labeled goat anti-rabbit IgG (green); smad2/3 was found in the cytoplasm of starved Capan-1 cells (a) and after treatment with B6.3 diabody (c). Smad2/3 was present in the nuclei in response to treatment with latent TGFβ 1 (b), a translocation that was inhibited by pre-treatment B6.3 diabody (d). TGFβ 1 was used as a positive control (e.f).
    Figure Legend Snippet: B6.3 diabody inhibited LAP-mediated Smad2/3 translocation to the nucleus in Capan-1 cells. Cells were incubated at 4°C in the presence of B6.3 diabody and then treated with LAP or TGFβ 1 (30 min, 37°C); Smad2/3 localization was assessed by confocal microscopy (40X) using rabbit anti-Smad2/3 followed by Alexa Fluor 488®-labeled goat anti-rabbit IgG (green); smad2/3 was found in the cytoplasm of starved Capan-1 cells (a) and after treatment with B6.3 diabody (c). Smad2/3 was present in the nuclei in response to treatment with latent TGFβ 1 (b), a translocation that was inhibited by pre-treatment B6.3 diabody (d). TGFβ 1 was used as a positive control (e.f).

    Techniques Used: Translocation Assay, Incubation, Confocal Microscopy, Labeling, Positive Control

    39) Product Images from "Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells"

    Article Title: Potent but transient immunosuppression of T-cells is a general feature of erythroid progenitor cells

    Journal: bioRxiv

    doi: 10.1101/2021.01.18.427109

    EPCs express ARG2 and have high levels of ROS a, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs (CD71 + TER119 + ) and RBCs (CD71 − TER119 + ) of control mice (n=6) and NHA (n=6) and HA-PHZ (n=6). Histograms show the representative fluorescence of CellROX Green – FITC in EPCs from the spleen of NHA mouse. P values calculated with unpaired t-test. b, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs, leukocytes (CD45 + ), T-cells (CD45 + CD3e + ), myeloid cells (CD45 + CD11b + ) (n=18). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. c, Percentages of ARG2 + EPCs in control mice (n=11), anemic mice (NHA, n=5; HA-PHZ, n=11; HA-TER119, n=11), neonatal mice (n=5), and isotype control-IgG-treated mice (control-IgG, n=7). d, Percentages of ARG1 + EPCs based on intracellular staining (n=5). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. e, Percentages of YFP + EPCs in reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=8, neonatal n=5, control-IgG n=4, HA-TER119 n=8). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. f, Mean Fluorescence Intensity (MFI) of YFP – FITC in EPCs of reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=4). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. g,h, Total arginase activity in EPCs lysates ( g, n=8) or in the supernatants from EPCs cultures ( h , n=8). P values calculated with one-way ANOVA with unpaired t-test. i, Percentages of ARG1 + EPCs isolated from the spleens of B6.129S4-Arg1 tm1Lky /J incubated with diluent or PHZ (100 μM for 24h) (n=3). P values calculated with one-way ANOVA with unpaired t-test. Data show means ± SD. Each point in a-i represents data from individual mice. n values are the numbers of mice used to obtain the data. The source data underlying Fig.3a-i are provided as a Source Data file.
    Figure Legend Snippet: EPCs express ARG2 and have high levels of ROS a, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs (CD71 + TER119 + ) and RBCs (CD71 − TER119 + ) of control mice (n=6) and NHA (n=6) and HA-PHZ (n=6). Histograms show the representative fluorescence of CellROX Green – FITC in EPCs from the spleen of NHA mouse. P values calculated with unpaired t-test. b, Mean Fluorescence Intensity (MFI) of CellROX Green – FITC in EPCs, leukocytes (CD45 + ), T-cells (CD45 + CD3e + ), myeloid cells (CD45 + CD11b + ) (n=18). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. c, Percentages of ARG2 + EPCs in control mice (n=11), anemic mice (NHA, n=5; HA-PHZ, n=11; HA-TER119, n=11), neonatal mice (n=5), and isotype control-IgG-treated mice (control-IgG, n=7). d, Percentages of ARG1 + EPCs based on intracellular staining (n=5). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. e, Percentages of YFP + EPCs in reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=8, neonatal n=5, control-IgG n=4, HA-TER119 n=8). P values calculated with one-way ANOVA with Dunnet’s post-hoc test and with unpaired t-test for HA-TER119. f, Mean Fluorescence Intensity (MFI) of YFP – FITC in EPCs of reporter B6.129S4-Arg1 tm1Lky /J mice (controls n=4, NHA n=8, HA-PHZ n=4). P values calculated with one-way ANOVA with Dunnet’s post-hoc test. g,h, Total arginase activity in EPCs lysates ( g, n=8) or in the supernatants from EPCs cultures ( h , n=8). P values calculated with one-way ANOVA with unpaired t-test. i, Percentages of ARG1 + EPCs isolated from the spleens of B6.129S4-Arg1 tm1Lky /J incubated with diluent or PHZ (100 μM for 24h) (n=3). P values calculated with one-way ANOVA with unpaired t-test. Data show means ± SD. Each point in a-i represents data from individual mice. n values are the numbers of mice used to obtain the data. The source data underlying Fig.3a-i are provided as a Source Data file.

    Techniques Used: Fluorescence, Mouse Assay, Staining, Activity Assay, Isolation, Incubation

    40) Product Images from "Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry"

    Article Title: Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

    Journal: Virology

    doi: 10.1016/j.virol.2018.05.009

    TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by DyLight 594-conjugated goat anti-rabbit IgG, Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).
    Figure Legend Snippet: TGEV infection induced EGFR internalization. (A) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. Then stained for fluorescence microscope using rabbit anti-EGFR pAb followed by DyLight 594-conjugated goat anti-rabbit IgG, Mock-infected cells served as controls. EGFR distribution was observed by confocal microscope. (B) Three-dimensional rendering of representative images obtained using Imaris 7.2 software. (C) IPEC-J2 cells were infected with TGEV (MOI = 2), and cultured for 1 h. The protein of the cell membrane was extracted. Cell membrane EGFR was analyzed by Westernblot using rabbit anti-EGFR pAb. (D) The ratio of EGFR to the mean of E-cadherin and GAPDH was normalized to control conditions. The data shown are the mean results ± SD, from three independent experiments. (scale bar = 20 µm).

    Techniques Used: Infection, Cell Culture, Staining, Fluorescence, Microscopy, Software

    TGEV infection causes the co-localization of APN and EGFR. IPEC-J2 cells were infected with TGEV (MOI = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-APN pAb, followed by DyLight 488-conjugated goat anti-mouse IgG and rabbit anti-p-EGFR mAb, followed by DyLight 594-conjugated goat anti-rabbit IgG. Mock-infected cells served as controls. The data shown are from two independent experiments.
    Figure Legend Snippet: TGEV infection causes the co-localization of APN and EGFR. IPEC-J2 cells were infected with TGEV (MOI = 2) and cultured for 30 min, then stained for fluorescence microscopy using mouse anti-APN pAb, followed by DyLight 488-conjugated goat anti-mouse IgG and rabbit anti-p-EGFR mAb, followed by DyLight 594-conjugated goat anti-rabbit IgG. Mock-infected cells served as controls. The data shown are from two independent experiments.

    Techniques Used: Infection, Cell Culture, Staining, Fluorescence, Microscopy

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    Staining:

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    Western Blot:

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  • 97
    Thermo Fisher goat anti rabbit igg alexa fluor 488
    Expression of integrin α 6 β 1 and stiffness of OPCs at different pH. ( a ) Expression level of integrin α 6 β 1 at different extracellular pH, evaluated by OPC immunostaining against α 6 β 1 (with <t>Alexa</t> <t>Fluor-488</t> fluorochrome) and analysis of cell fluorescence using flow cytometry (BD LSR Fortessa). Data are geometric mean fluorescence intensities averaged over three experiments, each conducted in triplicate, and presented relative to value obtained for pH 7.0. No statistical difference was observed between any pH conditions. ( b ) Cell stiffness at pH 6.0 and 7.0, evaluated using AFM-enabled nanoindentation. Data are mean of Young's elastic modulus measured for 15 cells per condition. No statistical difference was observed between pH 6.0 and 7.0. Error bars are SEM. Colors correspond to cell media pH.
    Goat Anti Rabbit Igg 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
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    99
    Thermo Fisher alexa 488 anti rabbit
    Efficacy of fluorophore inactivation and preservation of tissue integrity. ( A ) Exemplary image of a human tonsil stained with PCNA-Alexa 488 that underwent 0, 15, 30 or 60 min of fluorophore inactivation. ( B ) Effect of bleaching duration on the distribution of anti-PCNA-Alexa 488 staining intensities for samples used in ( A ). The distribution is computed from mean values for the fluorescence intensities across all cells in the image that were successfully segmented. The gray band denotes the range of background florescence intensities (below 6.2 in log scale). ( C ) Effect of bleaching duration on mean intensity for nine antibodies conjugated to Alexa fluor 488, efluor 570 or Alexa fluor 647. Intensities were determined as in ( B ). The gray band denotes the range of background florescence intensities. ( D ) Impact of t-CyCIF cycle number on tissue integrity for four exemplary tissue cores. Nuclei present in the first cycle are labeled in red and those present after the 10th cycle are in green. The numbers at the bottom of the images represent nuclear counts in cycle 1 (red) and cycle 10 (green), respectively. ( E ) Impact of t-CyCIF cycle number on the integrity of a TMA containing 48 biopsies obtained from 16 different healthy and tumor tissues (see Materials and methods for TMA details) stained with 10 rounds of t-CyCIF. The number of nuclei remaining in each core was computed relative to the starting value; small fluctuations in cell count explain values  >  1.0 and arise from errors in image segmentation. Data for six different breast cores is shown to the right. ( F ) Nuclear staining of a melanoma specimen subjected to 20 cycles of t-CyCIF emphasizes the preservation of tissue integrity (22 ± 4%). ( G ) Selected images of the specimen in ( F ) from cycles 0, 5, 15 and 20.
    Alexa 488 Anti Rabbit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti rabbit alexaflour 488 secondary igg
    Expression of HA-tagged tetherin proteins. Human and Pteropus alecto tetherin isoforms A, B, and C in HEK293T cells. Tetherin localisation in cells was detected using anti-HA-tag rabbit monoclonal Ig and anti-rabbit <t>AlexaFlour</t> 488 secondary Ig. Nuclei are blue stained with Hoescht. Fixed and permeabilised cells were imaged on Nikon AR1 confocal microscope. HA, haemagglutinin.
    Anti Rabbit Alexaflour 488 Secondary Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of integrin α 6 β 1 and stiffness of OPCs at different pH. ( a ) Expression level of integrin α 6 β 1 at different extracellular pH, evaluated by OPC immunostaining against α 6 β 1 (with Alexa Fluor-488 fluorochrome) and analysis of cell fluorescence using flow cytometry (BD LSR Fortessa). Data are geometric mean fluorescence intensities averaged over three experiments, each conducted in triplicate, and presented relative to value obtained for pH 7.0. No statistical difference was observed between any pH conditions. ( b ) Cell stiffness at pH 6.0 and 7.0, evaluated using AFM-enabled nanoindentation. Data are mean of Young's elastic modulus measured for 15 cells per condition. No statistical difference was observed between pH 6.0 and 7.0. Error bars are SEM. Colors correspond to cell media pH.

    Journal: PLoS ONE

    Article Title: Extracellular Acidic pH Inhibits Oligodendrocyte Precursor Viability, Migration, and Differentiation

    doi: 10.1371/journal.pone.0076048

    Figure Lengend Snippet: Expression of integrin α 6 β 1 and stiffness of OPCs at different pH. ( a ) Expression level of integrin α 6 β 1 at different extracellular pH, evaluated by OPC immunostaining against α 6 β 1 (with Alexa Fluor-488 fluorochrome) and analysis of cell fluorescence using flow cytometry (BD LSR Fortessa). Data are geometric mean fluorescence intensities averaged over three experiments, each conducted in triplicate, and presented relative to value obtained for pH 7.0. No statistical difference was observed between any pH conditions. ( b ) Cell stiffness at pH 6.0 and 7.0, evaluated using AFM-enabled nanoindentation. Data are mean of Young's elastic modulus measured for 15 cells per condition. No statistical difference was observed between pH 6.0 and 7.0. Error bars are SEM. Colors correspond to cell media pH.

    Article Snippet: Secondary antibodies included goat anti-mouse IgM Alexa Fluor 488 (Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen), and rabbit anti-rat IgG Alexa Fluor 488 (Invitrogen).

    Techniques: Expressing, Immunostaining, Fluorescence, Flow Cytometry, Cytometry

    The N1FR2011N-Vpu blocks anterograde transport of tetherin. ( A–D ) The nuclei of HT1080 cells, grown on coverslips, were co-microinjected with an expression plasmid encoding human tetherin with an internal HA tag together with vectors encoding either GFP ( A ) or C-terminally AU1 tagged Vpus: NL4-3 ( B ), N1FR2011 ( C ) and YBF30 ( D ). ∼200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2 or 6 hours and then fixed, permeabilized and stained with an anti-HA mAb followed by an Alexa 568-conjugated secondary a (red staining) to detect newly synthesized tetherin together with an anti-AU1 rab followed by Alexa 488-conjugated secondary antibody (green staining) to detect newly synthesized Vpu proteins. Microphotographs shown are representative for three independent experiments. Cell circumferences are indicated. Scale bars: 10 µm. ( E ) Stainings were categorized into cells, which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or cells with an exclusively intracellular staining (“intracellular”). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments.

    Journal: PLoS Pathogens

    Article Title: Human Tetherin Exerts Strong Selection Pressure on the HIV-1 Group N Vpu Protein

    doi: 10.1371/journal.ppat.1003093

    Figure Lengend Snippet: The N1FR2011N-Vpu blocks anterograde transport of tetherin. ( A–D ) The nuclei of HT1080 cells, grown on coverslips, were co-microinjected with an expression plasmid encoding human tetherin with an internal HA tag together with vectors encoding either GFP ( A ) or C-terminally AU1 tagged Vpus: NL4-3 ( B ), N1FR2011 ( C ) and YBF30 ( D ). ∼200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2 or 6 hours and then fixed, permeabilized and stained with an anti-HA mAb followed by an Alexa 568-conjugated secondary a (red staining) to detect newly synthesized tetherin together with an anti-AU1 rab followed by Alexa 488-conjugated secondary antibody (green staining) to detect newly synthesized Vpu proteins. Microphotographs shown are representative for three independent experiments. Cell circumferences are indicated. Scale bars: 10 µm. ( E ) Stainings were categorized into cells, which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or cells with an exclusively intracellular staining (“intracellular”). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments.

    Article Snippet: Newly synthesized Vpu molecules fused to AU-1 were detected using rabbit anti-AU1 (Covance) and anti-rabbit Alexa 488 secondary antibody (Invitrogen).

    Techniques: Expressing, Plasmid Preparation, Staining, Synthesized

    Efficacy of fluorophore inactivation and preservation of tissue integrity. ( A ) Exemplary image of a human tonsil stained with PCNA-Alexa 488 that underwent 0, 15, 30 or 60 min of fluorophore inactivation. ( B ) Effect of bleaching duration on the distribution of anti-PCNA-Alexa 488 staining intensities for samples used in ( A ). The distribution is computed from mean values for the fluorescence intensities across all cells in the image that were successfully segmented. The gray band denotes the range of background florescence intensities (below 6.2 in log scale). ( C ) Effect of bleaching duration on mean intensity for nine antibodies conjugated to Alexa fluor 488, efluor 570 or Alexa fluor 647. Intensities were determined as in ( B ). The gray band denotes the range of background florescence intensities. ( D ) Impact of t-CyCIF cycle number on tissue integrity for four exemplary tissue cores. Nuclei present in the first cycle are labeled in red and those present after the 10th cycle are in green. The numbers at the bottom of the images represent nuclear counts in cycle 1 (red) and cycle 10 (green), respectively. ( E ) Impact of t-CyCIF cycle number on the integrity of a TMA containing 48 biopsies obtained from 16 different healthy and tumor tissues (see Materials and methods for TMA details) stained with 10 rounds of t-CyCIF. The number of nuclei remaining in each core was computed relative to the starting value; small fluctuations in cell count explain values  >  1.0 and arise from errors in image segmentation. Data for six different breast cores is shown to the right. ( F ) Nuclear staining of a melanoma specimen subjected to 20 cycles of t-CyCIF emphasizes the preservation of tissue integrity (22 ± 4%). ( G ) Selected images of the specimen in ( F ) from cycles 0, 5, 15 and 20.

    Journal: eLife

    Article Title: Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes

    doi: 10.7554/eLife.31657

    Figure Lengend Snippet: Efficacy of fluorophore inactivation and preservation of tissue integrity. ( A ) Exemplary image of a human tonsil stained with PCNA-Alexa 488 that underwent 0, 15, 30 or 60 min of fluorophore inactivation. ( B ) Effect of bleaching duration on the distribution of anti-PCNA-Alexa 488 staining intensities for samples used in ( A ). The distribution is computed from mean values for the fluorescence intensities across all cells in the image that were successfully segmented. The gray band denotes the range of background florescence intensities (below 6.2 in log scale). ( C ) Effect of bleaching duration on mean intensity for nine antibodies conjugated to Alexa fluor 488, efluor 570 or Alexa fluor 647. Intensities were determined as in ( B ). The gray band denotes the range of background florescence intensities. ( D ) Impact of t-CyCIF cycle number on tissue integrity for four exemplary tissue cores. Nuclei present in the first cycle are labeled in red and those present after the 10th cycle are in green. The numbers at the bottom of the images represent nuclear counts in cycle 1 (red) and cycle 10 (green), respectively. ( E ) Impact of t-CyCIF cycle number on the integrity of a TMA containing 48 biopsies obtained from 16 different healthy and tumor tissues (see Materials and methods for TMA details) stained with 10 rounds of t-CyCIF. The number of nuclei remaining in each core was computed relative to the starting value; small fluctuations in cell count explain values  >  1.0 and arise from errors in image segmentation. Data for six different breast cores is shown to the right. ( F ) Nuclear staining of a melanoma specimen subjected to 20 cycles of t-CyCIF emphasizes the preservation of tissue integrity (22 ± 4%). ( G ) Selected images of the specimen in ( F ) from cycles 0, 5, 15 and 20.

    Article Snippet: Indirect immunofluorescence was performed using secondary antibodies conjugated with Alexa-647 anti-Mouse (Invitrogen, Cat. A-21236), Alexa-555 anti-Rat (Invitrogen, Cat. A-21434) and Alexa-488 anti-Rabbit (Invitrogen, Cat. A-11034).

    Techniques: Preserving, Staining, Fluorescence, Labeling, Cell Counting

    Expression of HA-tagged tetherin proteins. Human and Pteropus alecto tetherin isoforms A, B, and C in HEK293T cells. Tetherin localisation in cells was detected using anti-HA-tag rabbit monoclonal Ig and anti-rabbit AlexaFlour 488 secondary Ig. Nuclei are blue stained with Hoescht. Fixed and permeabilised cells were imaged on Nikon AR1 confocal microscope. HA, haemagglutinin.

    Journal: bioRxiv

    Article Title: Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

    doi: 10.1101/2020.04.08.031203

    Figure Lengend Snippet: Expression of HA-tagged tetherin proteins. Human and Pteropus alecto tetherin isoforms A, B, and C in HEK293T cells. Tetherin localisation in cells was detected using anti-HA-tag rabbit monoclonal Ig and anti-rabbit AlexaFlour 488 secondary Ig. Nuclei are blue stained with Hoescht. Fixed and permeabilised cells were imaged on Nikon AR1 confocal microscope. HA, haemagglutinin.

    Article Snippet: Cells were subsequently stained with anti-rabbit AlexaFlour 488 secondary IgG (Thermo) diluted in 0.2% Triton X-100, 3% BSA in PBS for 20 min at room temperature.

    Techniques: Expressing, Staining, Microscopy