α bungarotoxin btx conjugated to tetramethylrhodamine isothiocyanate  (Thermo Fisher)


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    Structured Review

    Thermo Fisher α bungarotoxin btx conjugated to tetramethylrhodamine isothiocyanate
    Fasudil increases endplate (EP) area in the tibialis anterior (TA) and transversus abdominis (TVA) muscles . Muscles were isolated from post-natal (P) day 21 untreated control littermates (n = 3), vehicle-treated Smn 2B/- (n = 4) and fasudil-treated Smn 2B/- mice (n = 3). ( A ) Representative images of TA and TVA EPs stained with <t>α-bungarotoxin</t> <t>(BTX).</t> Scale bars = 25 μm (TA) and 30 μm (TVA). ( B ) Quantification of EP area shows that fasudil-treated Smn 2B/- TA and TVA muscles display significantly larger EPs when compared to vehicle-treated Smn 2B/- muscles. (*** P
    α Bungarotoxin Btx Conjugated To Tetramethylrhodamine Isothiocyanate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 2928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α bungarotoxin btx conjugated to tetramethylrhodamine isothiocyanate - by Bioz Stars, 2020-03
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    Images

    1) Product Images from "Fasudil improves survival and promotes skeletal muscle development in a mouse model of spinal muscular atrophy"

    Article Title: Fasudil improves survival and promotes skeletal muscle development in a mouse model of spinal muscular atrophy

    Journal: BMC Medicine

    doi: 10.1186/1741-7015-10-24

    Fasudil increases endplate (EP) area in the tibialis anterior (TA) and transversus abdominis (TVA) muscles . Muscles were isolated from post-natal (P) day 21 untreated control littermates (n = 3), vehicle-treated Smn 2B/- (n = 4) and fasudil-treated Smn 2B/- mice (n = 3). ( A ) Representative images of TA and TVA EPs stained with α-bungarotoxin (BTX). Scale bars = 25 μm (TA) and 30 μm (TVA). ( B ) Quantification of EP area shows that fasudil-treated Smn 2B/- TA and TVA muscles display significantly larger EPs when compared to vehicle-treated Smn 2B/- muscles. (*** P
    Figure Legend Snippet: Fasudil increases endplate (EP) area in the tibialis anterior (TA) and transversus abdominis (TVA) muscles . Muscles were isolated from post-natal (P) day 21 untreated control littermates (n = 3), vehicle-treated Smn 2B/- (n = 4) and fasudil-treated Smn 2B/- mice (n = 3). ( A ) Representative images of TA and TVA EPs stained with α-bungarotoxin (BTX). Scale bars = 25 μm (TA) and 30 μm (TVA). ( B ) Quantification of EP area shows that fasudil-treated Smn 2B/- TA and TVA muscles display significantly larger EPs when compared to vehicle-treated Smn 2B/- muscles. (*** P

    Techniques Used: Isolation, Mouse Assay, Staining

    2) Product Images from "Microsatellite instability at a tetranucleotide repeat in type I endometrial carcinoma"

    Article Title: Microsatellite instability at a tetranucleotide repeat in type I endometrial carcinoma

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-27-88

    Examples of type I endometrial carcinoma with (A) loss of hMSH2 protein expression, (B) preservation of hMSH2 protein expression, (C) loss of hMLH1 protein expression, and (D) preservation of hMLH1 protein expression . Positive expression for hMSH2 or hMSH1 is noted in non-tumor cells. (A-D, X200).
    Figure Legend Snippet: Examples of type I endometrial carcinoma with (A) loss of hMSH2 protein expression, (B) preservation of hMSH2 protein expression, (C) loss of hMLH1 protein expression, and (D) preservation of hMLH1 protein expression . Positive expression for hMSH2 or hMSH1 is noted in non-tumor cells. (A-D, X200).

    Techniques Used: Expressing, Preserving

    3) Product Images from "?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling"

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010184

    N-terminally unphoshorylated β-catenin is a minor, nuclear form of β-catenin. A) Immunofluorescence of SW480 cells for total β-catenin or ABC (active β-catenin). Both monoclonal (mAb) and polyclonal (pAb) antibodies against total β-catenin produce a pan-cellular staining, while ABC predominantly stains nuclei. B) SW480 lysate (20 or 60 µg) was compared to known quantities of purified GST-β-catenin to estimate the relative abundance of total β-catenin to ABC. ABC is approximately 100 times less abundant than total β-catenin. Bars, 10 µm.
    Figure Legend Snippet: N-terminally unphoshorylated β-catenin is a minor, nuclear form of β-catenin. A) Immunofluorescence of SW480 cells for total β-catenin or ABC (active β-catenin). Both monoclonal (mAb) and polyclonal (pAb) antibodies against total β-catenin produce a pan-cellular staining, while ABC predominantly stains nuclei. B) SW480 lysate (20 or 60 µg) was compared to known quantities of purified GST-β-catenin to estimate the relative abundance of total β-catenin to ABC. ABC is approximately 100 times less abundant than total β-catenin. Bars, 10 µm.

    Techniques Used: Immunofluorescence, Staining, Purification

    β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.
    Figure Legend Snippet: β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.

    Techniques Used: Isolation, Affinity Precipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunofluorescence, Lysis, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    N-terminally unphosphorylated β-catenin appears highly sensitive to cadherin expression. A) Control (i. and iii.) and E-cadherin-restored SW480 cells (ii. and iv.) were stained with antibodies against total β-catenin or ABC. Note that ABC appears selectively recruited to sites of cell-cell contact upon cadherin expression relative to the total pool of β-catenin. B) A detergent-free cytosolic fraction from SW480/E-cadherin cells was subjected to gel filtration chromatography. Peak fractions are marked with arrows. The presence of E-cadherin reduces the abundance of monomeric ABC relative to control cells (compare to Fig. 2A ). C) Detergent-free preparations of membrane and cytosolic fractions isolated from control and E-cadherin-restored SW480 cells. Note that E-cadherin appears to selectively recruit ABC to the membrane, leaving a portion of total β-catenin in the cytosol. A non-specific band (*) recognized by ABC [47] serves as a loading control. Bars, 10 µm.
    Figure Legend Snippet: N-terminally unphosphorylated β-catenin appears highly sensitive to cadherin expression. A) Control (i. and iii.) and E-cadherin-restored SW480 cells (ii. and iv.) were stained with antibodies against total β-catenin or ABC. Note that ABC appears selectively recruited to sites of cell-cell contact upon cadherin expression relative to the total pool of β-catenin. B) A detergent-free cytosolic fraction from SW480/E-cadherin cells was subjected to gel filtration chromatography. Peak fractions are marked with arrows. The presence of E-cadherin reduces the abundance of monomeric ABC relative to control cells (compare to Fig. 2A ). C) Detergent-free preparations of membrane and cytosolic fractions isolated from control and E-cadherin-restored SW480 cells. Note that E-cadherin appears to selectively recruit ABC to the membrane, leaving a portion of total β-catenin in the cytosol. A non-specific band (*) recognized by ABC [47] serves as a loading control. Bars, 10 µm.

    Techniques Used: Expressing, Staining, Filtration, Chromatography, Isolation

    Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.
    Figure Legend Snippet: Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.

    Techniques Used: shRNA, Filtration, Chromatography, Purification, Fractionation, Labeling, Immunoprecipitation, Binding Assay

    N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).
    Figure Legend Snippet: N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).

    Techniques Used: Incubation, Binding Assay, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    β-catenin phosphorylated at T41/S45 is spatially uncoupled from β-catenin phosphorylated at S33/37/T41. SW480 cells were co-stained with antibodies against β-catenin phosphorylated at S33/37/T41 or T41/S45 and ABC. Merged images reveal that phospho-S33/37/T41 appears excluded from the nucleus, while phospho-T41/S45 is largely nuclear, as is ABC. Bars, 10 µm.
    Figure Legend Snippet: β-catenin phosphorylated at T41/S45 is spatially uncoupled from β-catenin phosphorylated at S33/37/T41. SW480 cells were co-stained with antibodies against β-catenin phosphorylated at S33/37/T41 or T41/S45 and ABC. Merged images reveal that phospho-S33/37/T41 appears excluded from the nucleus, while phospho-T41/S45 is largely nuclear, as is ABC. Bars, 10 µm.

    Techniques Used: Staining

    4) Product Images from "Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder"

    Article Title: Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder

    Journal: Scientific Reports

    doi: 10.1038/srep36110

    Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p
    Figure Legend Snippet: Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p

    Techniques Used: Expressing

    Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p
    Figure Legend Snippet: Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p

    Techniques Used: Staining, TUNEL Assay

    5) Product Images from "Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation"

    Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2009.09.021

    Confocal images of GFP expression from polymer transfected primary myoblasts (× 200). Control: non-treated primary myoblasts. Positive control: bPEI (25kDa)/pCMV-GFP at w/w ratio of 0.6:1. a–d: poly(CBA-DAH)/pCMV-GFP transfected primary
    Figure Legend Snippet: Confocal images of GFP expression from polymer transfected primary myoblasts (× 200). Control: non-treated primary myoblasts. Positive control: bPEI (25kDa)/pCMV-GFP at w/w ratio of 0.6:1. a–d: poly(CBA-DAH)/pCMV-GFP transfected primary

    Techniques Used: Expressing, Transfection, Positive Control

    6) Product Images from "Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2"

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    Journal: Veterinary Research

    doi: 10.1051/vetres/2010048

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )
    Figure Legend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Techniques Used: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing

    7) Product Images from "Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production"

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    Journal: Nature Communications

    doi: 10.1038/ncomms7765

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P
    Figure Legend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing

    8) Product Images from "Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis"

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    Journal: Journal of Proteome Research

    doi: 10.1021/pr200918f

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).
    Figure Legend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Techniques Used: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    9) Product Images from "B7x/B7-H4 modulates the adaptive immune response and ameliorates renal injury in antibody-mediated nephritis"

    Article Title: B7x/B7-H4 modulates the adaptive immune response and ameliorates renal injury in antibody-mediated nephritis

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12452

    Exacerbated infiltration of immune cells, immunoglobulin (Ig)G deposition and inflammatory mediator expression in kidneys of B7x −/− mice post-nephrotoxic serum (NTS). (a,b) Immunohistochemical analysis of infiltrating CD3 + T cells and
    Figure Legend Snippet: Exacerbated infiltration of immune cells, immunoglobulin (Ig)G deposition and inflammatory mediator expression in kidneys of B7x −/− mice post-nephrotoxic serum (NTS). (a,b) Immunohistochemical analysis of infiltrating CD3 + T cells and

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry

    10) Product Images from "Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses"

    Article Title: Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

    Journal: Retrovirology

    doi: 10.1186/1742-4690-5-2

    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.
    Figure Legend Snippet: Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Techniques Used: Expressing

    11) Product Images from "Functional test of PCDHB11, the most human-specific neuronal surface protein"

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein

    Journal: BMC Evolutionary Biology

    doi: 10.1186/s12862-016-0652-x

    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Figure Legend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Techniques Used: Expressing, Transfection, Staining

    12) Product Images from "Characterizing the Role of Cell-Wall ?-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37"

    Article Title: Characterizing the Role of Cell-Wall ?-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021394

    Binding of LL-37 to C. albicans cell-wall proteins. (A) Flow cytometry showing that BA-LL37 bound to C. albicans . Cells were treated with 1 mg/ml proteinase K (lower panels) or were not treated (upper panels) prior to incubation with 10 µg BA-LL37. The fluorescence intensity (FL1-H) of SA-DTAF that was associated with the cells via binding to BA-LL37 was measured to determine the amount of BA-LL37 bound to cells. The results are representative of two independent experiments that gave similar results. (B) Extracts prepared by fractionation of C. albicans cell-wall proteins using β-ME and β-glucanase. The proteins in the extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were probed with BA-LL37 and visualized with HRP–conjugated streptavidin. Arrows indicate the three major cell wall proteins bound by LL-37. The positions and values of molecular mass standards are indicated. Data are representative of three independent experiments that gave similar results.
    Figure Legend Snippet: Binding of LL-37 to C. albicans cell-wall proteins. (A) Flow cytometry showing that BA-LL37 bound to C. albicans . Cells were treated with 1 mg/ml proteinase K (lower panels) or were not treated (upper panels) prior to incubation with 10 µg BA-LL37. The fluorescence intensity (FL1-H) of SA-DTAF that was associated with the cells via binding to BA-LL37 was measured to determine the amount of BA-LL37 bound to cells. The results are representative of two independent experiments that gave similar results. (B) Extracts prepared by fractionation of C. albicans cell-wall proteins using β-ME and β-glucanase. The proteins in the extracts were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were probed with BA-LL37 and visualized with HRP–conjugated streptavidin. Arrows indicate the three major cell wall proteins bound by LL-37. The positions and values of molecular mass standards are indicated. Data are representative of three independent experiments that gave similar results.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Incubation, Fluorescence, Fractionation, SDS Page

    13) Product Images from "Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells"

    Article Title: Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells

    Journal: Journal of medical entomology

    doi:

    VCAM-1 expression by endothelial cells in response to varying doses of C. arcuatus and T. putrescentiae extracts. Cells were challenged for 6, 12, or 24 h with extract alone or along with TNF α . (Online figure in color.)
    Figure Legend Snippet: VCAM-1 expression by endothelial cells in response to varying doses of C. arcuatus and T. putrescentiae extracts. Cells were challenged for 6, 12, or 24 h with extract alone or along with TNF α . (Online figure in color.)

    Techniques Used: Expressing

    14) Product Images from "Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly"

    Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.198853

    Caveolin-1 interacts with the RSV G and M protein complex on the surface of infected cells. (A) Immunoprecipitations of RSV G, M and M2-1 proteins from HeLa whole-cell lysates of mock-infected or RSV-infected cells (22 hpi). Immunoprecipitates were probed for G protein (top panel), M protein (middle panel) and caveolin-1 (bottom panel). Caveolin-1 protein was measured by densitometry (bottom graph). (B) Streptavidin–HRP blot of lysates of surface-biotinylated HeLa cells. M, mock infected; I, RSV infected. (C) Streptavidin–HRP blot of immunoprecipitates from lysates shown in B, using antibodies against RSV G protein or caveolin-1. Representative data of two independent experiments are shown.
    Figure Legend Snippet: Caveolin-1 interacts with the RSV G and M protein complex on the surface of infected cells. (A) Immunoprecipitations of RSV G, M and M2-1 proteins from HeLa whole-cell lysates of mock-infected or RSV-infected cells (22 hpi). Immunoprecipitates were probed for G protein (top panel), M protein (middle panel) and caveolin-1 (bottom panel). Caveolin-1 protein was measured by densitometry (bottom graph). (B) Streptavidin–HRP blot of lysates of surface-biotinylated HeLa cells. M, mock infected; I, RSV infected. (C) Streptavidin–HRP blot of immunoprecipitates from lysates shown in B, using antibodies against RSV G protein or caveolin-1. Representative data of two independent experiments are shown.

    Techniques Used: Infection

    15) Product Images from "TRPC3 Activation by Erythropoietin Is Modulated by TRPC6"

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804734200

    Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.
    Figure Legend Snippet: Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

    Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.
    Figure Legend Snippet: Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

    16) Product Images from "Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells"

    Article Title: Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040030

    Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity. A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 µg) or LFt (20 µg) were incubated with 10 µg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 µM ZnCl 2 . Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p
    Figure Legend Snippet: Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity. A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 µg) or LFt (20 µg) were incubated with 10 µg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 µM ZnCl 2 . Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p

    Techniques Used: Recombinant, Activity Assay, Binding Assay, Purification, Incubation, MTT Assay, Standard Deviation

    17) Product Images from "Estradiol induces JNK-dependent apoptosis in glioblastoma cells"

    Article Title: Estradiol induces JNK-dependent apoptosis in glioblastoma cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2011.385

    Estradiol inhibited cell proliferation in C6 and T98G cells. Cells were pretreated with SP600125 (20 μM) or vehicle (control) for 30 min, and incubated for 18 h in the presence or absence of estradiol (20 μM) under low growth-stimulated conditions. BrdU incorporation in C6 cells (upper panel) and PCNA expression analysis in T98G cells (lower panel) were carried out. Images were captured using a brightfield microscope at a magnification of ×600.
    Figure Legend Snippet: Estradiol inhibited cell proliferation in C6 and T98G cells. Cells were pretreated with SP600125 (20 μM) or vehicle (control) for 30 min, and incubated for 18 h in the presence or absence of estradiol (20 μM) under low growth-stimulated conditions. BrdU incorporation in C6 cells (upper panel) and PCNA expression analysis in T98G cells (lower panel) were carried out. Images were captured using a brightfield microscope at a magnification of ×600.

    Techniques Used: Incubation, BrdU Incorporation Assay, Expressing, Microscopy

    18) Product Images from "Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules"

    Article Title: Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.017

    Expression of various envelope proteins on transfected cells and lentiviral vectors. (A) 293T cells transfected with control vector (black line) or envelope protein expression vectors (red line) were stained with anti-Sindbis virus antibody or biotinylated FITC and analyzed by flow cytometry. Flow cytometric profiles and mean fluorescence intensities of staining with anti-Sindbis virus envelope protein antibody and biotinylated FITC are shown. (B) Western blotting analysis of lentiviral vectors pseudotyped with: ① 2.2 1L1L, ② E2 71 AV, ③ E2 71 STAV, ④ E2 71 eMA, ⑤ E2 71 mSAH, or ⑥ 2.2. The same amounts of vectors (110 ng p24) were subjected to SDS-PAGE. After western blotting, the blotted membranes were stained with anti-Sindbis virus antibody, biotinylated HRP, or rabbit IgG-conjugated HRP.
    Figure Legend Snippet: Expression of various envelope proteins on transfected cells and lentiviral vectors. (A) 293T cells transfected with control vector (black line) or envelope protein expression vectors (red line) were stained with anti-Sindbis virus antibody or biotinylated FITC and analyzed by flow cytometry. Flow cytometric profiles and mean fluorescence intensities of staining with anti-Sindbis virus envelope protein antibody and biotinylated FITC are shown. (B) Western blotting analysis of lentiviral vectors pseudotyped with: ① 2.2 1L1L, ② E2 71 AV, ③ E2 71 STAV, ④ E2 71 eMA, ⑤ E2 71 mSAH, or ⑥ 2.2. The same amounts of vectors (110 ng p24) were subjected to SDS-PAGE. After western blotting, the blotted membranes were stained with anti-Sindbis virus antibody, biotinylated HRP, or rabbit IgG-conjugated HRP.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Western Blot, SDS Page

    19) Product Images from "Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly"

    Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.198853

    Caveolin-1 is recruited to RSV filaments in an actin-dependent manner. (A,B) RSV-infected HeLa cells were treated at 14 hpi with 500 nM cytochalasin D (A2,B) or left untreated (A1). Cells were fixed at 20 hpi and stained with antibodies against caveolin-1 and G protein, and phalloidin–FITC. Confocal micrographs are shown. (A1) RSV filaments are aligned along actin fibers. (A2) Partial disruption of the actin network with cytochalasin D causes distortion and aggregation of RSV filaments. Arrows indicate virus filaments (B) RSV-infected and cytochalasin-treated HeLa cells showing complete disruption of the actin cytoskeleton. Note the lack of RSV filaments and the loss of colocalization between caveolin-1 (red arrows) and G protein (green arrows). Representative data of two independent experiments are shown. Scale bars: 10 µm.
    Figure Legend Snippet: Caveolin-1 is recruited to RSV filaments in an actin-dependent manner. (A,B) RSV-infected HeLa cells were treated at 14 hpi with 500 nM cytochalasin D (A2,B) or left untreated (A1). Cells were fixed at 20 hpi and stained with antibodies against caveolin-1 and G protein, and phalloidin–FITC. Confocal micrographs are shown. (A1) RSV filaments are aligned along actin fibers. (A2) Partial disruption of the actin network with cytochalasin D causes distortion and aggregation of RSV filaments. Arrows indicate virus filaments (B) RSV-infected and cytochalasin-treated HeLa cells showing complete disruption of the actin cytoskeleton. Note the lack of RSV filaments and the loss of colocalization between caveolin-1 (red arrows) and G protein (green arrows). Representative data of two independent experiments are shown. Scale bars: 10 µm.

    Techniques Used: Infection, Staining

    20) Product Images from "Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide"

    Article Title: Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide

    Journal: Molecular and Cellular Biology

    doi:

    C 2 -ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C 2 -ceramide (C2) (100 μM) or C 2 -dihydroceramide (C 2 H 2 ) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with streptavidin-HRP and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of
    Figure Legend Snippet: C 2 -ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C 2 -ceramide (C2) (100 μM) or C 2 -dihydroceramide (C 2 H 2 ) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with streptavidin-HRP and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of

    Techniques Used: Translocation Assay, Immunofluorescence

    21) Product Images from "Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation *"

    Article Title: Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M111.009241

    Immunofluorescence and immunoblotting analysis of selected nucleolar proteins. A , WS1 cells were treated with UV (35 J/m 2 ) and IR (10 Gy) and fixed after 16 h. Cells were stained for AATF, GNL3 (nucleostemin), DDX56, and Ku70 proteins and imaged using
    Figure Legend Snippet: Immunofluorescence and immunoblotting analysis of selected nucleolar proteins. A , WS1 cells were treated with UV (35 J/m 2 ) and IR (10 Gy) and fixed after 16 h. Cells were stained for AATF, GNL3 (nucleostemin), DDX56, and Ku70 proteins and imaged using

    Techniques Used: Immunofluorescence, Staining

    22) Product Images from "Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington's Disease Knock-In Mice"

    Article Title: Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington's Disease Knock-In Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044273

    Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B. Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old HdhQ111 /+ mice with deletion of Msh2 in MSNs. A. Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice ( Msh2 +/+ CAG 113, Msh2Δ / Δ CAG 112, Msh2−/− CAG 113). B. Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of Msh2 genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and Msh2 genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P
    Figure Legend Snippet: Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B. Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old HdhQ111 /+ mice with deletion of Msh2 in MSNs. A. Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice ( Msh2 +/+ CAG 113, Msh2Δ / Δ CAG 112, Msh2−/− CAG 113). B. Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of Msh2 genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and Msh2 genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P

    Techniques Used: Mutagenesis, Immunostaining, Mouse Assay, Staining

    23) Product Images from "CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction"

    Article Title: CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction

    Journal:

    doi: 10.1016/j.immuni.2008.08.018

    In vivo co-localization of Foxp3 + T regs and mast cells and in vitro impairment of IgE-mediated degranulation of BMMCs by T regs
    Figure Legend Snippet: In vivo co-localization of Foxp3 + T regs and mast cells and in vitro impairment of IgE-mediated degranulation of BMMCs by T regs

    Techniques Used: In Vivo, In Vitro

    24) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    25) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    26) Product Images from "PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity"

    Article Title: PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028611

    Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.
    Figure Legend Snippet: Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.

    Techniques Used: Modification, Binding Assay, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay

    The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.
    Figure Legend Snippet: The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.

    Techniques Used: Cell Culture, Incubation, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Concentration Assay, Ex Vivo

    Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P
    Figure Legend Snippet: Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P

    Techniques Used: Modification, Cell Culture, Incubation, Functional Assay, Enzyme-linked Immunosorbent Assay

    27) Product Images from "TRPC3 Activation by Erythropoietin Is Modulated by TRPC6"

    Article Title: TRPC3 Activation by Erythropoietin Is Modulated by TRPC6

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804734200

    Interaction of TRPC3/TRPC6 chimeras with Epo-R. A, HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6C. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NRS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibody. Representative results of three similar experiments are shown. B, HEK 293T cells were transfected with Epo-R and FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N. Lysates were immunoprecipitated with anti-FLAG-agarose, anti-Epo-R, or normal rabbit serum. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R antibodies. Representative results of three experiments are shown. C, densitometry was used to quantitate Epo-R, V5-TRPC3, FLAG-TRPC6, and chimeric channel bands from three experiments using transfected HEK 293T cells. The Epo-R to V5-TRPC3 or V5-TRPC3-C6C ratio or Epo-R to FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N ratio was calculated and normalized to V5-TRPC3 or FLAG-TRPC6 to allow comparison between experiments. The mean normalized ratio ± S.E. was determined for three separate experiments. The Epo-R/V5-TRPC3-C6C ratio was significantly less than the Epo-R/V5-TRPC3 ratio ( * , p ≤ 0.01), and the Epo-R/FLAG-TRPC6-C3C ratio was significantly greater than the Epo-R/FLAG-TRPC6 or FLAG-TRPC6-C3N ratio ( ** , *** , p ≤ 0.001).
    Figure Legend Snippet: Interaction of TRPC3/TRPC6 chimeras with Epo-R. A, HEK 293T cells were transfected ( Tx'd ) with Epo-R and V5-TRPC3 or V5-TRPC3-C6C. Lysates were immunoprecipitated ( IP ) with anti-V5, anti-Epo-R antibodies, or normal rabbit serum ( NRS ). Western blots ( WB ) of lysates and immunoprecipitates were probed with anti-V5-HRP or anti-Epo-R and appropriate secondary antibody. Representative results of three similar experiments are shown. B, HEK 293T cells were transfected with Epo-R and FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N. Lysates were immunoprecipitated with anti-FLAG-agarose, anti-Epo-R, or normal rabbit serum. Western blots of lysates and immunoprecipitates were probed with anti-FLAG or anti-Epo-R antibodies. Representative results of three experiments are shown. C, densitometry was used to quantitate Epo-R, V5-TRPC3, FLAG-TRPC6, and chimeric channel bands from three experiments using transfected HEK 293T cells. The Epo-R to V5-TRPC3 or V5-TRPC3-C6C ratio or Epo-R to FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N ratio was calculated and normalized to V5-TRPC3 or FLAG-TRPC6 to allow comparison between experiments. The mean normalized ratio ± S.E. was determined for three separate experiments. The Epo-R/V5-TRPC3-C6C ratio was significantly less than the Epo-R/V5-TRPC3 ratio ( * , p ≤ 0.01), and the Epo-R/FLAG-TRPC6-C3C ratio was significantly greater than the Epo-R/FLAG-TRPC6 or FLAG-TRPC6-C3N ratio ( ** , *** , p ≤ 0.001).

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.
    Figure Legend Snippet: Modulation of membrane insertion of TRPC3 by Epo detected by cell surface biotinylation. HEK 293T cells transfected ( Tx'd ) with Epo-R and V5-TRPC3 without ( A and B ) or with FLAG-TRPC6 ( C and D ) were stimulated with 40 units/ml Epo. Biotinylation of cell surface proteins was performed, and V5-TRPC3 immunoprecipitated ( IP ) from lysates with anti-V5 antibody. Western blots ( WB ) were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-V5-HRP to detect total V5-TRPC3. Representative results of Western blots from four experiments are shown in A and C . Biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0, 1, 5, 10, and 20 min from four experiments are shown ( B and D ). * indicates a significant difference in the ratio compared with time 0 ( p ≤ 0.02). E, Epo-stimulated cell surface expression of endogenous TRPC3 was examined using BFU-E-derived erythroblasts at day 10 of methylcellulose culture (two experiments) or erythroblasts from phase II day 8 of liquid culture (one experiment). Cells were stimulated with 40 units/ml Epo for 0 or 5 min and biotinylated, and TRPC3 was immunoprecipitated from lysates with anti-TRPC3 antibody. Western blots were probed with streptavidin-HRP to detect biotinylated TRPC3 and anti-TRPC3 to detect total TRPC3. A representative result of three Western blots is shown. F, biotinylated and total TRPC3 bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. of the biotinylated/total TRPC3 ratios at 0 and 5 min from the three experiments are shown. No significant difference in the ratio at 5 min compared with time 0 was detected.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

    Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.
    Figure Legend Snippet: Plasma membrane insertion of TRPC3/TRPC3 chimeras detected with cell surface biotinylation. Cell surface biotinylation was performed with HEK 293T cells expressing V5-TRPC3, V5-TRPC3-C6C, V5-TRPC3-C6C1, V5-TRPC3-C6C2, FLAG-TRPC6, FLAG-TRPC6-C3C, or FLAG-TRPC6-C3N and Epo-R. Lysates were prepared, and immunoprecipitation ( IP ) performed with anti-V5 antibody or anti-FLAG-agarose. Western blotting ( WB ) was performed on immunoprecipitation pellets with streptavidin-HRP to detect biotinylation and either anti-V5-HRP to detect total TRPC3 chimeras or anti-TRPC6 or anti-TRPC3-N antibodies to detect total TRPC6 chimeras. Representative results of two experiments are shown. Tx'd , transfected.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Transfection

    28) Product Images from "?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling"

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010184

    β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.
    Figure Legend Snippet: β-catenin phosphorylated at S552 or S675 localizes to cell contacts and associates with E-cadherin. A) Cadherin-free β-catenin was isolated from an SW480 lysate by affinity precipitation with GST-ICAT, as previously described [48] . LC-MS/MS analysis identified S552 and S675 as two phosphorylation sites in β-catenin. Peptide abundance is plotted as a function of mass/charge (m/z). Identified phospho-sites are shown in red. B) Immunofluorescence of SW480 cells with antibodies to total β-catenin and phospho-S552 or -S675 reveals that phospho-S552 appears punctate, while phospho-S675 and total β-catenin localize uniformly to sites of cell-cell contact. C) Detergent-free lysis of membrane and cytosolic fractions from SW480/E-cad cells. D) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells. Note that phospho-S552 or -S675 float with cadherins. E) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection with streptavidin-HRP reveals that phospho-S552 and -S675 coimmunoprecipitate with a cell surface protein the same size as E-cadherin. Western blot for total β-catenin demonstrates immunoprecipitation efficiency. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad). An LRP6 immunoprecipitation was also performed and neither phospho-S552, -S675 nor N-terminal phospho-forms of β-catenin coimmunoprecipitate with a surface protein of this size. Bars, 10 µm.

    Techniques Used: Isolation, Affinity Precipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunofluorescence, Lysis, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).
    Figure Legend Snippet: N-terminally phosphorylated β-catenin is largely not associated with E-cadherin. A) SW480 cell lysates were sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. Non-binding lane reflects 5% of the total unbound fraction. Note that while ABC can be affinity precipitated by GST-cadherin, β-catenin phosphorylated at S45, T41/S45, and S33/37/T41 bind to a lesser extent. B) Sucrose gradient density centrifugation of the detergent-free membrane preparation from SW480/E-cadherin cells reveals that N-terminally phosphorylated β-catenin does not appreciably co-fractionate (i.e., float) with cadherins. C) Immunoprecipitation of Axin or E-cadherin from SW480/E-cadherin lysates reveals that β-catenin phosphorylated at S33/37/T41 does not associate with E-cadherin. D) Cell surface biotinylation of SW480/E-cadherin cells followed by immunoprecipitation with the indicated antibodies and detection by streptavidin-HRP reveals that ABC coimmunoprecipitates with a cell surface protein the same size as E-cadherin, while β-catenin phosphorylated at S33/37/T41 does not. Western blot analysis for total β-catenin confirms that the same amount of β-catenin was immunopreciptated with antibodies against T41/S45 and S33/37/T41. Mouse IgG (mIgG) and rabbit IgG (rIgG) controls are shown, as well as a positive control for E-cadherin (IP: E-cad).

    Techniques Used: Incubation, Binding Assay, Centrifugation, Immunoprecipitation, Western Blot, Positive Control

    29) Product Images from "PKC? Promotes HuD-Mediated Neprilysin mRNA Stability and Enhances Neprilysin-Induced A? Degradation in Brain Neurons"

    Article Title: PKC? Promotes HuD-Mediated Neprilysin mRNA Stability and Enhances Neprilysin-Induced A? Degradation in Brain Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097756

    PKCε activation recovers NEP membrane localization inhibited by oligomeric Aβ peptides. A , Cells were untreated or treated with oligomeric Aβ (Abeta; 1 µM) or Abeta+bryostatin (Bryo, 1 nM), or pre-incubated with Ro 32-0432 (Ro, 2 µM) for 30 min before Abeta+Bryo treatment for 1 hr, and then used for biotin-labeling. Phosphorylated and biotinylated NEP proteins were detected by immunoblot from immunoprecipitated NEP protein and were compared (Mean ± SEM of the three independent experiments, *P
    Figure Legend Snippet: PKCε activation recovers NEP membrane localization inhibited by oligomeric Aβ peptides. A , Cells were untreated or treated with oligomeric Aβ (Abeta; 1 µM) or Abeta+bryostatin (Bryo, 1 nM), or pre-incubated with Ro 32-0432 (Ro, 2 µM) for 30 min before Abeta+Bryo treatment for 1 hr, and then used for biotin-labeling. Phosphorylated and biotinylated NEP proteins were detected by immunoblot from immunoprecipitated NEP protein and were compared (Mean ± SEM of the three independent experiments, *P

    Techniques Used: Activation Assay, Incubation, Labeling, Immunoprecipitation

    Activated PKCε increases phosphorylation and membrane localization of NEP. A , Cells were untreated or treated with bryostatin (Bryo, 0.5 nM) or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with Bryo for 1 hr, lysed, and then used for immunoprecipitation using NEP antibody. Immunocomplexes were further analyzed to detect phosphorylated and biotinylated NEP protein in immunoblot analyses after normalization for NEP protein level (Mean ± SEM, *P
    Figure Legend Snippet: Activated PKCε increases phosphorylation and membrane localization of NEP. A , Cells were untreated or treated with bryostatin (Bryo, 0.5 nM) or pre-treated with Ro 32-0432 (Ro, 2 µM) for 30 min and then treated with Bryo for 1 hr, lysed, and then used for immunoprecipitation using NEP antibody. Immunocomplexes were further analyzed to detect phosphorylated and biotinylated NEP protein in immunoblot analyses after normalization for NEP protein level (Mean ± SEM, *P

    Techniques Used: Immunoprecipitation

    30) Product Images from "Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus"

    Article Title: Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018477

    The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.
    Figure Legend Snippet: The enzyme-labeled LD5 exhibits enhanced binding activities for IgM and IgA. The binding activities of horseradish peroxidase (HRP)-labeled LD5 (HRP-LD5) or HRP-conjugated goat anti-human polyclonal antibodies (HRP-goat anti-human PcAb) to coated hIgM (A), hIgG (B) or hIgA (C) were examined by ELISA. The coating buffer was used as solvent control.

    Techniques Used: Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay

    31) Product Images from "VRC01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu-BLT mice"

    Article Title: VRC01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu-BLT mice

    Journal: Archives of virology

    doi: 10.1007/s00705-016-2942-4

    Expression and purification of VRC01 IgG antibody. Polyacrylamide gel with Coomassie blue staining. The antibody was purified using protein A as described in Materials and methods. A total of 8 µg of purified protein was loaded onto this SDS-PAGE
    Figure Legend Snippet: Expression and purification of VRC01 IgG antibody. Polyacrylamide gel with Coomassie blue staining. The antibody was purified using protein A as described in Materials and methods. A total of 8 µg of purified protein was loaded onto this SDS-PAGE

    Techniques Used: Expressing, Purification, Staining, SDS Page

    VRC01 neutralizing activity against inoculating virus
    Figure Legend Snippet: VRC01 neutralizing activity against inoculating virus

    Techniques Used: Activity Assay

    Kinetics of VRC01 antibody in HIV- 1-naïve hu-BLT mice. VRC01 concentrations in sera of three hu-BLT mice after subcutaneous injection of 400 µg VRC01 IgG antibody
    Figure Legend Snippet: Kinetics of VRC01 antibody in HIV- 1-naïve hu-BLT mice. VRC01 concentrations in sera of three hu-BLT mice after subcutaneous injection of 400 µg VRC01 IgG antibody

    Techniques Used: Mouse Assay, Injection

    Comparison of the neutralizing activity of VRC01 against HIV-1 Ada and five transmitted/founder (T/F) HIV-1 viruses. VRC01 IgG antibody inhibited the infection with T/F HIV-1 viruses (TRJO, REJO, SUMA, WITO, and RHPA) and HIV-1 Ada in vitro . The neutralization
    Figure Legend Snippet: Comparison of the neutralizing activity of VRC01 against HIV-1 Ada and five transmitted/founder (T/F) HIV-1 viruses. VRC01 IgG antibody inhibited the infection with T/F HIV-1 viruses (TRJO, REJO, SUMA, WITO, and RHPA) and HIV-1 Ada in vitro . The neutralization

    Techniques Used: Activity Assay, Infection, In Vitro, Neutralization

    Protection of VRC01 against vaginal transmission of HIV-1. VRC01 IgG antibody (20 µg/g body weight) was administered subcutaneously to three hu-BLT mice (VRC01 treatment group), while the same dosage of human IgG was injected into another three
    Figure Legend Snippet: Protection of VRC01 against vaginal transmission of HIV-1. VRC01 IgG antibody (20 µg/g body weight) was administered subcutaneously to three hu-BLT mice (VRC01 treatment group), while the same dosage of human IgG was injected into another three

    Techniques Used: Transmission Assay, Mouse Assay, Injection

    Protection of VRC01 against rectal transmission of HIV-1. VRC01 IgG antibody (20 µg/g body weight) was administered subcutaneously to six hu-BLT mice (VRC01 treatment group), and human IgG was injected into another six mice (control group), and
    Figure Legend Snippet: Protection of VRC01 against rectal transmission of HIV-1. VRC01 IgG antibody (20 µg/g body weight) was administered subcutaneously to six hu-BLT mice (VRC01 treatment group), and human IgG was injected into another six mice (control group), and

    Techniques Used: Transmission Assay, Mouse Assay, Injection

    32) Product Images from "Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba"

    Article Title: Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2014.47.10.192

    Cry4Ba binding to the larval midgut epithelium and toxin proteolytic processing in the larvae. (A) Larval tissue sections were covered by Cry4Ba wild type (Cry4Ba) and mutants (indicated). A bound toxin was detected with antibodies C4bPAbs, this was followed by secondary antibodies; biotin conjugated anti-mouse IgG /streptavidin-HRP (see Methods). The dark-brown color represents toxin binding at BBM (arrow heads) from 3-independent experiments. A control is a larval section which was reacted with antibodies, but without toxin reaction (control). (B) Western blot analysis of a toxin associated with PM or gut in the larvae treated with Cry4Ba wild type (lane 2) and R158A (lane 3) with Cry4Ba specific antibodies C4bPAbs. Antibodies show negative reactivity with proteins which were isolated from the PMs and guts of normal larvae (lane 1). (C) Reactivity of the antibodies C4bPAbs with Y170A protein associated with gut (lane 1) and PM (lane 2). The 47 kDa and 18-20 kDa of the trypsin activated Cry4Ba in vitro (B-lane 4, C-lane 3) were used as positive controls.
    Figure Legend Snippet: Cry4Ba binding to the larval midgut epithelium and toxin proteolytic processing in the larvae. (A) Larval tissue sections were covered by Cry4Ba wild type (Cry4Ba) and mutants (indicated). A bound toxin was detected with antibodies C4bPAbs, this was followed by secondary antibodies; biotin conjugated anti-mouse IgG /streptavidin-HRP (see Methods). The dark-brown color represents toxin binding at BBM (arrow heads) from 3-independent experiments. A control is a larval section which was reacted with antibodies, but without toxin reaction (control). (B) Western blot analysis of a toxin associated with PM or gut in the larvae treated with Cry4Ba wild type (lane 2) and R158A (lane 3) with Cry4Ba specific antibodies C4bPAbs. Antibodies show negative reactivity with proteins which were isolated from the PMs and guts of normal larvae (lane 1). (C) Reactivity of the antibodies C4bPAbs with Y170A protein associated with gut (lane 1) and PM (lane 2). The 47 kDa and 18-20 kDa of the trypsin activated Cry4Ba in vitro (B-lane 4, C-lane 3) were used as positive controls.

    Techniques Used: Binding Assay, Western Blot, Isolation, In Vitro

    33) Product Images from "Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors"

    Article Title: Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18429-0

    Fibronectin and fibulin-1 deposition by immortalised asthmatic and non-asthmatic ASM cells. FN and FBLN-1 deposition from immortalised non-asthmatic (n = 5) and asthmatic (n = 6) ASM cells after 48 hrs ( a and b respectively) or 72 hrs ( c and d respectively) in response to treatment with 10 ng/ml TGF-β. All data are presented as the mean +/− SEM. Statistical analysis via a two way ANOVA with Sidak’s multiple comparisons test was performed to test differences between control and treatment groups and immortalised asthmatic and non-asthmatic ASM cells. Significance as a result of treatment is represented by **p
    Figure Legend Snippet: Fibronectin and fibulin-1 deposition by immortalised asthmatic and non-asthmatic ASM cells. FN and FBLN-1 deposition from immortalised non-asthmatic (n = 5) and asthmatic (n = 6) ASM cells after 48 hrs ( a and b respectively) or 72 hrs ( c and d respectively) in response to treatment with 10 ng/ml TGF-β. All data are presented as the mean +/− SEM. Statistical analysis via a two way ANOVA with Sidak’s multiple comparisons test was performed to test differences between control and treatment groups and immortalised asthmatic and non-asthmatic ASM cells. Significance as a result of treatment is represented by **p

    Techniques Used:

    34) Product Images from "Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses"

    Article Title: Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-293

    CD99 is recruited into the IS and lipid rafts . (A) DDAO-SE pre-labeled Jurkat T cells were incubated with SEB-loaded Raji B cells. Cells were then plated onto a cover slip, fixed, stained with anti-CD99 mAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, TRITC-phalloidin for F-actin and Hoechst 33258 for the nucleus. The stained cells were visualized by confocal fluorescence microscopy. The green and yellow images represent the localization and distribution of CD99 and F-actin, respectively. The nuclei and Jurkat cells are shown in blue and red, respectively. (B) Jurkat cells were homogenized to isolate cell membranes, which were then solubilized in lipid raft preserving detergent 1% Brij-98 or lipid raft disrupting detergent 1% Lauryl maltoside (LM) and subjected to gel filtration on Sepharose 4B. The obtained fractions were analyzed by Western immunoblotting using anti-CD99 and anti-Lck mAbs.
    Figure Legend Snippet: CD99 is recruited into the IS and lipid rafts . (A) DDAO-SE pre-labeled Jurkat T cells were incubated with SEB-loaded Raji B cells. Cells were then plated onto a cover slip, fixed, stained with anti-CD99 mAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, TRITC-phalloidin for F-actin and Hoechst 33258 for the nucleus. The stained cells were visualized by confocal fluorescence microscopy. The green and yellow images represent the localization and distribution of CD99 and F-actin, respectively. The nuclei and Jurkat cells are shown in blue and red, respectively. (B) Jurkat cells were homogenized to isolate cell membranes, which were then solubilized in lipid raft preserving detergent 1% Brij-98 or lipid raft disrupting detergent 1% Lauryl maltoside (LM) and subjected to gel filtration on Sepharose 4B. The obtained fractions were analyzed by Western immunoblotting using anti-CD99 and anti-Lck mAbs.

    Techniques Used: Labeling, Incubation, Staining, Fluorescence, Microscopy, Preserving, Filtration, Western Blot

    35) Product Images from "Point-of-care test for cervical cancer in LMICs"

    Article Title: Point-of-care test for cervical cancer in LMICs

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7709

    Scheme of enzyme-enhanced LFIC for VCP detection Biotinylated gold nanoparticles tethered with streptavidin-bearing HRP at the capture site in the lateral flow strip to generate a signal upon interaction with TMB.
    Figure Legend Snippet: Scheme of enzyme-enhanced LFIC for VCP detection Biotinylated gold nanoparticles tethered with streptavidin-bearing HRP at the capture site in the lateral flow strip to generate a signal upon interaction with TMB.

    Techniques Used: Flow Cytometry, Stripping Membranes

    36) Product Images from "Effective screen for amyloid ? aggregation inhibitor using amyloid ?-conjugated gold nanoparticles"

    Article Title: Effective screen for amyloid ? aggregation inhibitor using amyloid ?-conjugated gold nanoparticles

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S15278

    Activity validation of an Aβ aggregation inhibitor TTR, using AuNP–Aβ42 precipitates. In inducing AuNP–Aβ42 aggregation, its coincubation with TTR prevented the formation of visible AuNP–Aβ42 precipitates and maintained the red color of the colloidal solution, as in the AuNP control A ). TTR reversed the significant decrease in optical density B ), and increase in ThT value C ) of AuNP–Aβ42, resulting from the inhibition of both Aβ aggregation and AuNP–Aβ42 and one-way ANOVA (* P
    Figure Legend Snippet: Activity validation of an Aβ aggregation inhibitor TTR, using AuNP–Aβ42 precipitates. In inducing AuNP–Aβ42 aggregation, its coincubation with TTR prevented the formation of visible AuNP–Aβ42 precipitates and maintained the red color of the colloidal solution, as in the AuNP control A ). TTR reversed the significant decrease in optical density B ), and increase in ThT value C ) of AuNP–Aβ42, resulting from the inhibition of both Aβ aggregation and AuNP–Aβ42 and one-way ANOVA (* P

    Techniques Used: Activity Assay, Inhibition

    Precipitation pattern of AuNP–Aβ42 incubated with sera from normal and AD patients. Images were taken before TEM analysis in Figure 6 . Abbreviations: AD, Alzheimer’s disease; Aβ42, amyloid β42; AuNP, gold nanoparticle; TEM, transmission electron microscopy.
    Figure Legend Snippet: Precipitation pattern of AuNP–Aβ42 incubated with sera from normal and AD patients. Images were taken before TEM analysis in Figure 6 . Abbreviations: AD, Alzheimer’s disease; Aβ42, amyloid β42; AuNP, gold nanoparticle; TEM, transmission electron microscopy.

    Techniques Used: Incubation, Transmission Electron Microscopy, Transmission Assay, Electron Microscopy

    Schematic of sequence of Aβ42 conjugation to AuNP surface via biotin–streptavidin interaction and formation of AuNP–Aβ42 precipitate by Aβ42 aggregation. Abbreviations: Aβ amyloid β Aβ42, amyloid β42; AuNP, gold nanoparticle.
    Figure Legend Snippet: Schematic of sequence of Aβ42 conjugation to AuNP surface via biotin–streptavidin interaction and formation of AuNP–Aβ42 precipitate by Aβ42 aggregation. Abbreviations: Aβ amyloid β Aβ42, amyloid β42; AuNP, gold nanoparticle.

    Techniques Used: Sequencing, Conjugation Assay

    Confirmation of Aβ42 conjugation to AuNP and induction of AuNP–Aβ42 precipitates by Aβ42 aggregation. A ) Verification of biotin–Aβ42 conjugation to streptavidin-AuNP by dot blot analysis. B ) After 48 hours incubation, visible AuNP–Aβ42 precipitates formed in the bottom of the tubes and the supernatant became clear; no precipitate was observed in the AuNP control. C ) Optical density of supernatant as a quantitative indicator of AuNP–Aβ42 precipitation (Paired t -test, *** P
    Figure Legend Snippet: Confirmation of Aβ42 conjugation to AuNP and induction of AuNP–Aβ42 precipitates by Aβ42 aggregation. A ) Verification of biotin–Aβ42 conjugation to streptavidin-AuNP by dot blot analysis. B ) After 48 hours incubation, visible AuNP–Aβ42 precipitates formed in the bottom of the tubes and the supernatant became clear; no precipitate was observed in the AuNP control. C ) Optical density of supernatant as a quantitative indicator of AuNP–Aβ42 precipitation (Paired t -test, *** P

    Techniques Used: Conjugation Assay, Dot Blot, Incubation

    TEM analysis of AuNP–Aβ42 precipitates. A ) AuNP–Aβ42 formed clusters with short interparticle distances, and AuNP underwent an even distribution of particles. The addition of free Aβ42 resulted in a similar pattern of aggregation as AuNP–Aβ42 but a greater interparticle distance and dark background staining. TTR blocked the formation of AuNP–Aβ42 aggregates and maintained the homogeneous dispersion of particles as evenly as the AuNP control. B ) Interparticle distances were measured and compared (*** P
    Figure Legend Snippet: TEM analysis of AuNP–Aβ42 precipitates. A ) AuNP–Aβ42 formed clusters with short interparticle distances, and AuNP underwent an even distribution of particles. The addition of free Aβ42 resulted in a similar pattern of aggregation as AuNP–Aβ42 but a greater interparticle distance and dark background staining. TTR blocked the formation of AuNP–Aβ42 aggregates and maintained the homogeneous dispersion of particles as evenly as the AuNP control. B ) Interparticle distances were measured and compared (*** P

    Techniques Used: Transmission Electron Microscopy, Staining

    Precipitation pattern of AuNP–Aβ42 with or without TTR or free Aβ42. Images were taken before the TEM analysis in Figure 5 . Abbreviations: Aβ42, amyloid β42; AuNP, gold nanoparticle; TEM, transmission electron microscopy; TTR, transtyretin.
    Figure Legend Snippet: Precipitation pattern of AuNP–Aβ42 with or without TTR or free Aβ42. Images were taken before the TEM analysis in Figure 5 . Abbreviations: Aβ42, amyloid β42; AuNP, gold nanoparticle; TEM, transmission electron microscopy; TTR, transtyretin.

    Techniques Used: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy

    TEM analysis of AuNP–Aβ42 incubated with blood-derived serum from normal and AD patients. Sera from normal individuals (n = 4) and AD patients (n = 4) were incubated with AuNP–Aβ42 during aggregation. AuNP–Aβ42 in AD patient serum showed aggregation and significantly shorter interparticle distances ( A right, B right, and C ) (*** P
    Figure Legend Snippet: TEM analysis of AuNP–Aβ42 incubated with blood-derived serum from normal and AD patients. Sera from normal individuals (n = 4) and AD patients (n = 4) were incubated with AuNP–Aβ42 during aggregation. AuNP–Aβ42 in AD patient serum showed aggregation and significantly shorter interparticle distances ( A right, B right, and C ) (*** P

    Techniques Used: Transmission Electron Microscopy, Incubation, Derivative Assay

    AuNP–Aβ42 precipitates in an Aβ42 concentration-dependent manner. Various concentrations of biotin–Aβ42 (0, 0.5, 1, 5, 10, 50, 100, and 300 μmol/L) were added to streptavidin-AuNP to determine the optimal concentration of Aβ42 conjugation. Visible precipitates developed at 50 μmol/L biotin–Aβ42 A ), accompanied by a decrease in optical density B ), and increase in ThT binding C ), indicating that 10–50 μmol/L is the optimal concentration of Aβ42 to saturate AuNP surfaces and induce AuNP–Aβ42 aggregation. Above 50 μmol/L, free Aβ42 reverses the optical density despite the increased ThT value. Abbreviations: Aβ42, amyloid β42; AuNP, gold nanoparticle; ThT, thioflavin T.
    Figure Legend Snippet: AuNP–Aβ42 precipitates in an Aβ42 concentration-dependent manner. Various concentrations of biotin–Aβ42 (0, 0.5, 1, 5, 10, 50, 100, and 300 μmol/L) were added to streptavidin-AuNP to determine the optimal concentration of Aβ42 conjugation. Visible precipitates developed at 50 μmol/L biotin–Aβ42 A ), accompanied by a decrease in optical density B ), and increase in ThT binding C ), indicating that 10–50 μmol/L is the optimal concentration of Aβ42 to saturate AuNP surfaces and induce AuNP–Aβ42 aggregation. Above 50 μmol/L, free Aβ42 reverses the optical density despite the increased ThT value. Abbreviations: Aβ42, amyloid β42; AuNP, gold nanoparticle; ThT, thioflavin T.

    Techniques Used: Concentration Assay, Conjugation Assay, Binding Assay

    Time-dependent aggregation and precipitation pattern of AuNP–Aβ42. Abbreviations: Aβ, amyloid β; Aβ42, amyloid β42; AuNP, gold nanoparticle; ThT, thioflavin T.
    Figure Legend Snippet: Time-dependent aggregation and precipitation pattern of AuNP–Aβ42. Abbreviations: Aβ, amyloid β; Aβ42, amyloid β42; AuNP, gold nanoparticle; ThT, thioflavin T.

    Techniques Used:

    37) Product Images from "Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator"

    Article Title: Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

    Journal:

    doi: 10.1042/BJ20061123

    CG7 increases the number of IR undergoing IRβ auto-phosphorylation in CHO/IR cells
    Figure Legend Snippet: CG7 increases the number of IR undergoing IRβ auto-phosphorylation in CHO/IR cells

    Techniques Used:

    Effect of CG7 and/or insulin washout on IRβ auto-phosphorylation in CHO/IR cells
    Figure Legend Snippet: Effect of CG7 and/or insulin washout on IRβ auto-phosphorylation in CHO/IR cells

    Techniques Used:

    Insulin-stimulated IRβ auto-phosphorylation in CHO/IR cells and effects of triterpenoids on IR
    Figure Legend Snippet: Insulin-stimulated IRβ auto-phosphorylation in CHO/IR cells and effects of triterpenoids on IR

    Techniques Used:

    38) Product Images from "Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses"

    Article Title: Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-293

    Co-immunoprecipitation of CD99 with other membrane proteins . Immunoprecipitation of biotinylated Jurkat cell lysates using mild detergent 1% Brij-58 (A) or raft-disrupting detergent 1% Lauryl maltoside (B). Cell lysates were precipitated using isotype matched control 4G2 mAb (lane 1) and anti-CD99 mAb MT99/3 (lane 2). Electrophoresis was performed under reducing conditions. The positions of molecular mass markers are indicated on the left in kDa. The positions of CD99 molecules are indicated.
    Figure Legend Snippet: Co-immunoprecipitation of CD99 with other membrane proteins . Immunoprecipitation of biotinylated Jurkat cell lysates using mild detergent 1% Brij-58 (A) or raft-disrupting detergent 1% Lauryl maltoside (B). Cell lysates were precipitated using isotype matched control 4G2 mAb (lane 1) and anti-CD99 mAb MT99/3 (lane 2). Electrophoresis was performed under reducing conditions. The positions of molecular mass markers are indicated on the left in kDa. The positions of CD99 molecules are indicated.

    Techniques Used: Immunoprecipitation, Electrophoresis

    39) Product Images from "Dax1 Binds to Oct3/4 and Inhibits Its Transcriptional Activity in Embryonic Stem Cells ▿Dax1 Binds to Oct3/4 and Inhibits Its Transcriptional Activity in Embryonic Stem Cells ▿ †"

    Article Title: Dax1 Binds to Oct3/4 and Inhibits Its Transcriptional Activity in Embryonic Stem Cells ▿Dax1 Binds to Oct3/4 and Inhibits Its Transcriptional Activity in Embryonic Stem Cells ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01863-08

    Nuclear localization of Dax1 is enhanced by Oct3/4. HeLa cells were transfected with GFP-Dax1 plus Myc-DsRed or GFP-Dax1 plus Myc-DsRed-Oct3/4. Cells were photographed 2 days after transfection. Bars, 50 μm.
    Figure Legend Snippet: Nuclear localization of Dax1 is enhanced by Oct3/4. HeLa cells were transfected with GFP-Dax1 plus Myc-DsRed or GFP-Dax1 plus Myc-DsRed-Oct3/4. Cells were photographed 2 days after transfection. Bars, 50 μm.

    Techniques Used: Transfection

    40) Product Images from "IL-12 p80-dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection"

    Article Title: IL-12 p80-dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02923.x

    Resistance to a lethal Sendai virus infection in the p80/p40 transgenic mice is independent of viral load. (a) Wild-type (WT; top) or p80/p40 Tg (bottom) littermates were inoculated with Sendai virus 50 000 egg infectious dose 50% (50 K) and day 5 post-inoculation lung sections were immunolabelled with anti-Sendai antibody (Ab) [detected with fluorescein isothiocyanate (FITC); green] and anti-CD68 Ab (detected with Alexa Fluor 555; red). Top and bottom rows are identical views photographed with a filter for the green channel (column 1), a filter for the red channel (column 2), and a merged image (column 3). Control immunoglobulin G (IgG) Ab gave no signal above background (not shown). Arrows indicate dual-labelled cells. Representative photomicrographs are shown ( n = 4). Bar, 20 μm. (b) Wild-type (WT) and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K and day 3, 5, 8 and 12 whole-lung homogenates were analysed for Sendai plaque-forming units (PFU)/g of lung tissue. Values represent mean ± standard deviation for duplicate samples ( n = 6–8). (c) Wild-type and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K or 5 K and whole-lung RNA from day 3, 5 and 8 post-inoculation was analysed for Sendai virus-specific and GAPDH RNA by one-step fluorogenic reverse transcriptase–polymerase chain reaction (RT-PCR). The mean of duplicate measurements of Sendai virus-specific RNA was normalized to GAPDH and reported as the Sendai to GAPDH ratio. A significant difference from WT is indicated (* P
    Figure Legend Snippet: Resistance to a lethal Sendai virus infection in the p80/p40 transgenic mice is independent of viral load. (a) Wild-type (WT; top) or p80/p40 Tg (bottom) littermates were inoculated with Sendai virus 50 000 egg infectious dose 50% (50 K) and day 5 post-inoculation lung sections were immunolabelled with anti-Sendai antibody (Ab) [detected with fluorescein isothiocyanate (FITC); green] and anti-CD68 Ab (detected with Alexa Fluor 555; red). Top and bottom rows are identical views photographed with a filter for the green channel (column 1), a filter for the red channel (column 2), and a merged image (column 3). Control immunoglobulin G (IgG) Ab gave no signal above background (not shown). Arrows indicate dual-labelled cells. Representative photomicrographs are shown ( n = 4). Bar, 20 μm. (b) Wild-type (WT) and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K and day 3, 5, 8 and 12 whole-lung homogenates were analysed for Sendai plaque-forming units (PFU)/g of lung tissue. Values represent mean ± standard deviation for duplicate samples ( n = 6–8). (c) Wild-type and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K or 5 K and whole-lung RNA from day 3, 5 and 8 post-inoculation was analysed for Sendai virus-specific and GAPDH RNA by one-step fluorogenic reverse transcriptase–polymerase chain reaction (RT-PCR). The mean of duplicate measurements of Sendai virus-specific RNA was normalized to GAPDH and reported as the Sendai to GAPDH ratio. A significant difference from WT is indicated (* P

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Standard Deviation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    41) Product Images from "Folate-targeted immunotherapy effectively treats established adjuvant and collagen-induced arthritis"

    Article Title: Folate-targeted immunotherapy effectively treats established adjuvant and collagen-induced arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar1944

    Comparison of the effects of various arthritis therapies on disease severity in rats with established adjuvant-induced arthritic (AIA). Rats were left either unimmunized or immunized with keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) in an equal volume of alum adjuvant to generate a high anti-FITC antibody titer. AIA was induced by the base-of-tail method, and 7 days after induction each therapy was initiated. The therapies were folate-FITC in non-immunized rats (folate-FITC; 30 nmole/kg per day, i.p.), non-targeted aminofluorescein (AF) in immunized rats (KLH-FITC + AF; 30 nmole/kg per day, i.p.), clodronate liposomes (CL; 3.6 mg/kg on days 8, 16, and 23, i.p.), celecoxib (20 mg/kg every other day, oral gavage), etanercept (4 mg/kg per day, i.p), anakinra (120 mg/kg per day, continuous infusion, Alzet osmotic pump), methotrexate (MTX; 0.75 mg/kg per week, i.p.), or folate-targeted immunotherapy (FTI) in immunized rats (KLH-FITC + folate-FITC [FTI]; 30 nmole/kg per day, i.p.). All groups were measured for arthritis symptoms by monitoring changes in ankle diameter (a) , bone and cartilage degradation (b) , uptake of EC20 in the spleen and liver (c) , and splenomegaly (d) . Data are representative of two independent experiments (mean ± standard deviation, n = 5 rats/group).
    Figure Legend Snippet: Comparison of the effects of various arthritis therapies on disease severity in rats with established adjuvant-induced arthritic (AIA). Rats were left either unimmunized or immunized with keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) in an equal volume of alum adjuvant to generate a high anti-FITC antibody titer. AIA was induced by the base-of-tail method, and 7 days after induction each therapy was initiated. The therapies were folate-FITC in non-immunized rats (folate-FITC; 30 nmole/kg per day, i.p.), non-targeted aminofluorescein (AF) in immunized rats (KLH-FITC + AF; 30 nmole/kg per day, i.p.), clodronate liposomes (CL; 3.6 mg/kg on days 8, 16, and 23, i.p.), celecoxib (20 mg/kg every other day, oral gavage), etanercept (4 mg/kg per day, i.p), anakinra (120 mg/kg per day, continuous infusion, Alzet osmotic pump), methotrexate (MTX; 0.75 mg/kg per week, i.p.), or folate-targeted immunotherapy (FTI) in immunized rats (KLH-FITC + folate-FITC [FTI]; 30 nmole/kg per day, i.p.). All groups were measured for arthritis symptoms by monitoring changes in ankle diameter (a) , bone and cartilage degradation (b) , uptake of EC20 in the spleen and liver (c) , and splenomegaly (d) . Data are representative of two independent experiments (mean ± standard deviation, n = 5 rats/group).

    Techniques Used: Standard Deviation

    Folate-targeted immunotherapy reduces folate receptor expressing macrophages in inflamed tissue of AIA rats. (a) The level of folate receptor (FR)+ activated macrophages in the limbs of non-immunized (Folate-fluorescein isothiocyanate [FITC]) and keyhole limpet hemocyanine (KLH)-FITC-immunized AIA rats (KLH-FITC + Folate-FITC [FTI]) 25 days after initiation of treatment with folate-FITC was determined 4 hours after i.p. injection of 0.5 mg EC20. Folate-FITC was administered at 375 nmole/kg twice a week until day 23, and estimates of FR + macrophage numbers were made by weighing and counting the limbs for EC20 radioactivity (± standard deviation for n = 5 rats/group; [folate-FITC] vs. [KLH-FITC + folate-FITC (FTI)], p
    Figure Legend Snippet: Folate-targeted immunotherapy reduces folate receptor expressing macrophages in inflamed tissue of AIA rats. (a) The level of folate receptor (FR)+ activated macrophages in the limbs of non-immunized (Folate-fluorescein isothiocyanate [FITC]) and keyhole limpet hemocyanine (KLH)-FITC-immunized AIA rats (KLH-FITC + Folate-FITC [FTI]) 25 days after initiation of treatment with folate-FITC was determined 4 hours after i.p. injection of 0.5 mg EC20. Folate-FITC was administered at 375 nmole/kg twice a week until day 23, and estimates of FR + macrophage numbers were made by weighing and counting the limbs for EC20 radioactivity (± standard deviation for n = 5 rats/group; [folate-FITC] vs. [KLH-FITC + folate-FITC (FTI)], p

    Techniques Used: Expressing, Injection, Radioactivity, Standard Deviation

    Effect of folate-targeted immunotherapy on joint inflammation and bone/cartilage destruction in AIA rats. Where indicated, AIA rats were immunized with a 1:1 suspension of keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) and TiterMax Gold adjuvant to generate a high anti-FITC antibody titer. On day 0, rats were induced to develop AIA via the footpad method. (a) Treatments began on the day of arthritis induction, and rats were given i.p. doses twice a week at 375 nmole folate-FITC/kg. Treatment groups included: AIA rats not immunized and not treated (No Treatment), non-immunized rats treated with folate-FITC (Folate-FITC), AIA rats immunized against fluorescein and treated with non-targeted aminofluorescein (KLH-FITC + AF), AIA rats immunized against fluorescein and treated with folic acid alone (KLH-FITC + Folate), AIA rats immunized against fluorescein but not further treated (KLH-FITC), AIA rats immunized against fluorescein and treated with folate-FITC (KLH-FITC + folate-FITC [FTI]), AIA rats treated on days 8, 16, and 23 with 3.6 mg/kg clodronate liposomes (CL), AIA rats treated with methotrexate (MTX; 0.75 mg/kg per week, i.p.), immunized healthy rats (KLH-FITC + Healthy), and untreated healthy rats (Healthy), respectively. Data are representative of two independent experiments ( n = 5 rats/group; [folate-FITC] vs. [KLH-FITC + folate-FITC (FTI)], p
    Figure Legend Snippet: Effect of folate-targeted immunotherapy on joint inflammation and bone/cartilage destruction in AIA rats. Where indicated, AIA rats were immunized with a 1:1 suspension of keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) and TiterMax Gold adjuvant to generate a high anti-FITC antibody titer. On day 0, rats were induced to develop AIA via the footpad method. (a) Treatments began on the day of arthritis induction, and rats were given i.p. doses twice a week at 375 nmole folate-FITC/kg. Treatment groups included: AIA rats not immunized and not treated (No Treatment), non-immunized rats treated with folate-FITC (Folate-FITC), AIA rats immunized against fluorescein and treated with non-targeted aminofluorescein (KLH-FITC + AF), AIA rats immunized against fluorescein and treated with folic acid alone (KLH-FITC + Folate), AIA rats immunized against fluorescein but not further treated (KLH-FITC), AIA rats immunized against fluorescein and treated with folate-FITC (KLH-FITC + folate-FITC [FTI]), AIA rats treated on days 8, 16, and 23 with 3.6 mg/kg clodronate liposomes (CL), AIA rats treated with methotrexate (MTX; 0.75 mg/kg per week, i.p.), immunized healthy rats (KLH-FITC + Healthy), and untreated healthy rats (Healthy), respectively. Data are representative of two independent experiments ( n = 5 rats/group; [folate-FITC] vs. [KLH-FITC + folate-FITC (FTI)], p

    Techniques Used:

    Effect of folate-targeted immunotherapy (FTI) on mice with established collagen-induced arthritis. Mice were immunized with keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) in an equal volume of TiterMax Gold adjuvant to generate a high anti-FITC antibody titer. Seven days after arthritis induction, when the average arthritis score had reached 7, mice were treated daily with 600 nmole/kg folate-FITC until day 38. (a) Differences between folate-FITC-treated and untreated mice were easily visualized. (b) Each limb was evaluated weekly by an individual blinded to the treatment groups and was assigned an arthritis score (see Methods and methods). (c) Bone erosion was analyzed on day 49 from radiographs by a radiologist blinded to the treatment groups. (d) Uptake of EC20 in both hind limbs was determined as a measure of activated macrophage accumulation on day 49, represented as percent injected dose of EC20 per gram hind limbs, for instance, %ID EC20/g hind limbs. (e) The change in weight of mice after arthritis induction was measured regularly.
    Figure Legend Snippet: Effect of folate-targeted immunotherapy (FTI) on mice with established collagen-induced arthritis. Mice were immunized with keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC) in an equal volume of TiterMax Gold adjuvant to generate a high anti-FITC antibody titer. Seven days after arthritis induction, when the average arthritis score had reached 7, mice were treated daily with 600 nmole/kg folate-FITC until day 38. (a) Differences between folate-FITC-treated and untreated mice were easily visualized. (b) Each limb was evaluated weekly by an individual blinded to the treatment groups and was assigned an arthritis score (see Methods and methods). (c) Bone erosion was analyzed on day 49 from radiographs by a radiologist blinded to the treatment groups. (d) Uptake of EC20 in both hind limbs was determined as a measure of activated macrophage accumulation on day 49, represented as percent injected dose of EC20 per gram hind limbs, for instance, %ID EC20/g hind limbs. (e) The change in weight of mice after arthritis induction was measured regularly.

    Techniques Used: Mouse Assay, Injection

    No indication of toxicity was detected in rats treated with folate-targeted immunotherapy for 15 weeks. Toxicity analysis associated with continuous treatment of healthy keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC)-immunized rats with folate-targeted immunotherapy (FTI) for 15 weeks. Non-immunized or KLH-FITC-immunized rats were treated with folate-FITC (300 nmole/kg, three times per week) and sacrificed at the indicated times. Serum was analyzed for indicators of kidney (creatinine) (a) and liver (aspartate aminotransferase [AST-SGOT]) (b) function. Tissue samples, including liver, kidney, brain, lungs, and bone marrow, were simultaneously submitted for histopathological analyses. No indication of toxicity was detected by either type of analysis (tissue section data not shown) (mean ± standard deviation, n = 8 rats/group).
    Figure Legend Snippet: No indication of toxicity was detected in rats treated with folate-targeted immunotherapy for 15 weeks. Toxicity analysis associated with continuous treatment of healthy keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC)-immunized rats with folate-targeted immunotherapy (FTI) for 15 weeks. Non-immunized or KLH-FITC-immunized rats were treated with folate-FITC (300 nmole/kg, three times per week) and sacrificed at the indicated times. Serum was analyzed for indicators of kidney (creatinine) (a) and liver (aspartate aminotransferase [AST-SGOT]) (b) function. Tissue samples, including liver, kidney, brain, lungs, and bone marrow, were simultaneously submitted for histopathological analyses. No indication of toxicity was detected by either type of analysis (tissue section data not shown) (mean ± standard deviation, n = 8 rats/group).

    Techniques Used: AST Assay, Standard Deviation

    Folate-targeted immunotherapy reduces activated macrophages systemically in adjuvant-induced arthritic rats. (a) Treatment schedule for FTI. (b) The level of activated macrophages in internal organs (left) and limbs (right) of non-immunized arthritic rats (upper panel) and keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC)-immunized arthritic rats (lower panel) 25 days after initiation of treatment with folate-FITC (375 nmole/kg twice a week until day 21) was imaged 4 hours after i.p. injection of 0.5 mg EC20. (c) Activated macrophage accumulation in the liver and spleen was evaluated by measuring EC20 uptake in healthy non-arthritic rats (Healthy), AIA rats immunized with KLH-FITC and left untreated (KLH-FITC), non-immunized rats injected i.p. with folate-FITC (375 nmole/kg twice a week), KLH-FITC-immunized AIA rats treated with folate-FITC (KLH-FITC + Folate-FITC [FTI], 375 nmole/kg twice a week), and AIA rats injected with clodronate liposomes ([CL]; 3.6 mg/kg on days 8, 16, and 21, i.p.). EC20 biodistribution was measured by removal of the indicated tissues, weighing and counting them for radioactivity; represented as percent injected dose of EC20 per gram tissue, for instance, %ID EC20/g tissue (mean ± standard deviation, n = 5 rats/group). Data are representative of three independent experiments. (c) folate-FITC versus KLH-FITC + folate-FITC (FTI), p
    Figure Legend Snippet: Folate-targeted immunotherapy reduces activated macrophages systemically in adjuvant-induced arthritic rats. (a) Treatment schedule for FTI. (b) The level of activated macrophages in internal organs (left) and limbs (right) of non-immunized arthritic rats (upper panel) and keyhole limpet hemocyanine-fluorescein isothiocyanate (KLH-FITC)-immunized arthritic rats (lower panel) 25 days after initiation of treatment with folate-FITC (375 nmole/kg twice a week until day 21) was imaged 4 hours after i.p. injection of 0.5 mg EC20. (c) Activated macrophage accumulation in the liver and spleen was evaluated by measuring EC20 uptake in healthy non-arthritic rats (Healthy), AIA rats immunized with KLH-FITC and left untreated (KLH-FITC), non-immunized rats injected i.p. with folate-FITC (375 nmole/kg twice a week), KLH-FITC-immunized AIA rats treated with folate-FITC (KLH-FITC + Folate-FITC [FTI], 375 nmole/kg twice a week), and AIA rats injected with clodronate liposomes ([CL]; 3.6 mg/kg on days 8, 16, and 21, i.p.). EC20 biodistribution was measured by removal of the indicated tissues, weighing and counting them for radioactivity; represented as percent injected dose of EC20 per gram tissue, for instance, %ID EC20/g tissue (mean ± standard deviation, n = 5 rats/group). Data are representative of three independent experiments. (c) folate-FITC versus KLH-FITC + folate-FITC (FTI), p

    Techniques Used: Injection, Radioactivity, Standard Deviation

    42) Product Images from "Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease"

    Article Title: Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease

    Journal: Glycobiology

    doi: 10.1093/glycob/cwn072

    Anti-Neu5Gc antibodies in normal humans are of broad and variable specificities. ( A ) Levels of anti-Neu5Gcα-PAA and anti-GM3(Neu5Gc) IgG antibodies in normal human sera ( n = 16) were quantified in triplicates by ELISA using Neu5Ac-glycans for
    Figure Legend Snippet: Anti-Neu5Gc antibodies in normal humans are of broad and variable specificities. ( A ) Levels of anti-Neu5Gcα-PAA and anti-GM3(Neu5Gc) IgG antibodies in normal human sera ( n = 16) were quantified in triplicates by ELISA using Neu5Ac-glycans for

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Human serum anti-Neu5Gc reactivity is selectively inhibited by 2- O -methyl-α-Neu5Gc and by Neu5Gc-bound to glycoproteins. ( A ) ELISA inhibition assay (EIA) of anti-Neu5Gcα2-6Lac- IgG antibodies in human serum S34 with either 2- O -methyl-α-Neu5Gc
    Figure Legend Snippet: Human serum anti-Neu5Gc reactivity is selectively inhibited by 2- O -methyl-α-Neu5Gc and by Neu5Gc-bound to glycoproteins. ( A ) ELISA inhibition assay (EIA) of anti-Neu5Gcα2-6Lac- IgG antibodies in human serum S34 with either 2- O -methyl-α-Neu5Gc

    Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition

    43) Product Images from "PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity"

    Article Title: PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028611

    PEGylated TNYL-RAW inhibits capillary-like tube formation in co-cultured HUVECs and EMCs. EMCs expressing EGFP and cultured in DMEM or complete Medium 200 and HUVECs labeled with CellTracker™ Orange were plated on Matrigel individually or mixed at a 1∶2 ratio and imaged 5 and 20 hours later. The effect of 5 µM PEG-TNYL-RAW or an equal amount of PEG control on tube formation was analyzed. The histogram shows average tube lengths quantified from 2–3 micrographs at 5 hours for EMCs and 20 hours for HUVECs and HUVEC + EMC co-cultures and normalized to the average for the PEG control. Error bars represent the standard error from tube length measured from 3–4 wells. Tube length in the presence of PEG-TNYL-RAW was compared to that in the presence of PEG by one-way ANOVA and Bonferroni's post test. ***P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits capillary-like tube formation in co-cultured HUVECs and EMCs. EMCs expressing EGFP and cultured in DMEM or complete Medium 200 and HUVECs labeled with CellTracker™ Orange were plated on Matrigel individually or mixed at a 1∶2 ratio and imaged 5 and 20 hours later. The effect of 5 µM PEG-TNYL-RAW or an equal amount of PEG control on tube formation was analyzed. The histogram shows average tube lengths quantified from 2–3 micrographs at 5 hours for EMCs and 20 hours for HUVECs and HUVEC + EMC co-cultures and normalized to the average for the PEG control. Error bars represent the standard error from tube length measured from 3–4 wells. Tube length in the presence of PEG-TNYL-RAW was compared to that in the presence of PEG by one-way ANOVA and Bonferroni's post test. ***P

    Techniques Used: Cell Culture, Expressing, Labeling

    Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.
    Figure Legend Snippet: Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.

    Techniques Used: Modification, Binding Assay, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay

    The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.
    Figure Legend Snippet: The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.

    Techniques Used: Cell Culture, Incubation, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Concentration Assay, Ex Vivo

    PEGylation increases the effectiveness of the TNYL-RAW peptide in inhibiting capillary-like tube formation by co-coltured HUVECs and EMCs. HUVECs labeled with CellTracker™ Orange and EMCs expressing EGFP were plated on Matrigel in complete Medium 200 at a 2∶1 ratio and imaged 15 hours later. The effect of different concentrations of PEG-TNYL-RAW or TNYL-RAW on tube formation was analyzed. The histograms show the average tube lengths for the different peptide treatments normalized to the average for the PEG or DMSO controls. Error bars represent the standard error from 3–4 wells. Tube lengths for PEG-TNYL-RAW or TNYL-RAW were compared to the PEG or DMSO control by one-way ANOVA and Dunnett's post test. **P
    Figure Legend Snippet: PEGylation increases the effectiveness of the TNYL-RAW peptide in inhibiting capillary-like tube formation by co-coltured HUVECs and EMCs. HUVECs labeled with CellTracker™ Orange and EMCs expressing EGFP were plated on Matrigel in complete Medium 200 at a 2∶1 ratio and imaged 15 hours later. The effect of different concentrations of PEG-TNYL-RAW or TNYL-RAW on tube formation was analyzed. The histograms show the average tube lengths for the different peptide treatments normalized to the average for the PEG or DMSO controls. Error bars represent the standard error from 3–4 wells. Tube lengths for PEG-TNYL-RAW or TNYL-RAW were compared to the PEG or DMSO control by one-way ANOVA and Dunnett's post test. **P

    Techniques Used: Labeling, Expressing

    PEGylated TNYL-RAW inhibits capillary sprouting in co-coltured HUVECs and EMCs. Collagen embedded spheroids generated with HUVECs expressing mCherry, EMCs expressing EGFP or a 1∶1 mixture of the two cell types were treated with 5 µM PEG-TNYL-RAW or PEG for 2 days. The number of sprouts and the cumulative sprout length in the HUVE + EMC spheroids were normalized to the average for the PEG control. The histogram shows averages from 40–45 spheroids ± SE. The values obtained for spheroids treated with PEG-TNYL-RAW were compared to those with PEG control by one-way ANOVA. ***P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits capillary sprouting in co-coltured HUVECs and EMCs. Collagen embedded spheroids generated with HUVECs expressing mCherry, EMCs expressing EGFP or a 1∶1 mixture of the two cell types were treated with 5 µM PEG-TNYL-RAW or PEG for 2 days. The number of sprouts and the cumulative sprout length in the HUVE + EMC spheroids were normalized to the average for the PEG control. The histogram shows averages from 40–45 spheroids ± SE. The values obtained for spheroids treated with PEG-TNYL-RAW were compared to those with PEG control by one-way ANOVA. ***P

    Techniques Used: Generated, Expressing

    Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P
    Figure Legend Snippet: Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P

    Techniques Used: Modification, Cell Culture, Incubation, Functional Assay, Enzyme-linked Immunosorbent Assay

    PEGylated TNYL-RAW inhibits tyrosine phosphorylation of EphB4 and ephrin-B2. (A) B16 melanoma cells pretreated with the indicated concentrations of PEG-TNYL-RAW or TNYL-RAW for 15 min or 24 hours were stimulated with 1.5 µg/ml preclustered ephrin-B2 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the peptide. EphB4 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (B) The inhibition curve shows the relative levels of EphB4 phosphorylation in the presence of different concentrations of PEG-TNYL-RAW, which were quantified from immunoblots and normalized to the amount of immunoprecipitated EphB4. Error bars represent the standard error from 3–6 experiments. (C) HUVEC and EMC lysates were probed for EphB4, ephrin-B2 (band at ∼45 Kd detected with a pan-ephrin-B antibody) and ß-actin as a loading control. It is not known why the ephrin-B2 band appears as a more prominent doublet in EMCs than HUVECs. (D) HUVECs and EMCs were cultured individually or mixed at a 1∶1 ratio in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. EphB4 immunoprecipitates were probed with an anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (E) HUVECs and EMCs, which express EGFP, were cultured at a 1∶1 ratio for 15 hours in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. The cells were then stimulated with 1.5 µg/ml preclustered EphB4 Fc or Fc as a control for 20 min in the continued presence of the peptide or PEG. The cells were stained for phospho-ephrin-B (red), which likely corresponds to the phosphorylated form of the EphB4 preferred ligand ephrin-B2, and nuclei were labeled with DAPI (blue). Scale bar = 50 µM. Fluorescence intensity from 6 micrographs per condition was quantified. The values obtained (expressed in arbitrary units ± standard error) are: PEG Fc, 24±2.6; PEG EphB4 Fc, 44±3.6; PEG-TNYL-RAW EphB4 Fc, 20±2.5. The fluorescence of cells treated with PEG-TNYL-RAW EphB4 Fc was significantly (P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits tyrosine phosphorylation of EphB4 and ephrin-B2. (A) B16 melanoma cells pretreated with the indicated concentrations of PEG-TNYL-RAW or TNYL-RAW for 15 min or 24 hours were stimulated with 1.5 µg/ml preclustered ephrin-B2 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the peptide. EphB4 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (B) The inhibition curve shows the relative levels of EphB4 phosphorylation in the presence of different concentrations of PEG-TNYL-RAW, which were quantified from immunoblots and normalized to the amount of immunoprecipitated EphB4. Error bars represent the standard error from 3–6 experiments. (C) HUVEC and EMC lysates were probed for EphB4, ephrin-B2 (band at ∼45 Kd detected with a pan-ephrin-B antibody) and ß-actin as a loading control. It is not known why the ephrin-B2 band appears as a more prominent doublet in EMCs than HUVECs. (D) HUVECs and EMCs were cultured individually or mixed at a 1∶1 ratio in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. EphB4 immunoprecipitates were probed with an anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (E) HUVECs and EMCs, which express EGFP, were cultured at a 1∶1 ratio for 15 hours in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. The cells were then stimulated with 1.5 µg/ml preclustered EphB4 Fc or Fc as a control for 20 min in the continued presence of the peptide or PEG. The cells were stained for phospho-ephrin-B (red), which likely corresponds to the phosphorylated form of the EphB4 preferred ligand ephrin-B2, and nuclei were labeled with DAPI (blue). Scale bar = 50 µM. Fluorescence intensity from 6 micrographs per condition was quantified. The values obtained (expressed in arbitrary units ± standard error) are: PEG Fc, 24±2.6; PEG EphB4 Fc, 44±3.6; PEG-TNYL-RAW EphB4 Fc, 20±2.5. The fluorescence of cells treated with PEG-TNYL-RAW EphB4 Fc was significantly (P

    Techniques Used: Inhibition, Western Blot, Immunoprecipitation, Cell Culture, Staining, Labeling, Fluorescence

    44) Product Images from "A Combination of Flt3 Ligand cDNA and CpG ODN as Nasal Adjuvant Elicits NALT Dendritic Cells for Prolonged Mucosal Immunity"

    Article Title: A Combination of Flt3 Ligand cDNA and CpG ODN as Nasal Adjuvant Elicits NALT Dendritic Cells for Prolonged Mucosal Immunity

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2008.06.091

    Immunofluorescence staining of CD11c + DCs in NALT. BALB/c mice were immunized with OVA and plasmid encoding Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN) ( A, B ), pFL ( C, D ), or CpG ODN ( E, F ) as nasal adjuvants. NALT taken from naïve mice were also stained as controls ( G, H ). Frozen sections of NALT were stained with biotin-conjugated, anti-CD11c (HL3) mAbs followed by HRP-conjugated streptavidin-Alexa Fluor 488®. The original magnification was × 40 ( A, C, E, G ) and ×100 ( B, D, F, H ). The picture is a typical example of results of immunofluorescence analysis of over 20 samples.
    Figure Legend Snippet: Immunofluorescence staining of CD11c + DCs in NALT. BALB/c mice were immunized with OVA and plasmid encoding Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN) ( A, B ), pFL ( C, D ), or CpG ODN ( E, F ) as nasal adjuvants. NALT taken from naïve mice were also stained as controls ( G, H ). Frozen sections of NALT were stained with biotin-conjugated, anti-CD11c (HL3) mAbs followed by HRP-conjugated streptavidin-Alexa Fluor 488®. The original magnification was × 40 ( A, C, E, G ) and ×100 ( B, D, F, H ). The picture is a typical example of results of immunofluorescence analysis of over 20 samples.

    Techniques Used: Immunofluorescence, Staining, Mouse Assay, Plasmid Preparation

    45) Product Images from "Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules"

    Article Title: Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

    Journal: Virology

    doi: 10.1016/j.virol.2018.09.017

    Site-specific biotinylation of the anti-hTfR1 antibody. (A) Schematic representation of conjugation of antibodies randomly biotinylated at lysine residues, the Fab fragment specifically biotinylated at C-terminal cysteines, and the biotinylated EGF. (B) ① disulfide bonds between heavy chains of anti-hTfR1 antibody are reduced by 2-mercaptethanolamine; ② the reduced cysteines were biotinylated with maleimide biotin; and ③ Fc regions of the biotinylated antibody are digested with pepsin. (C) ① Biotinylated anti-hTfR1 antibody; and ② the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody subjected to SDS-PAGE, followed by SyproRuby staining and western blotting, using HRP-conjugated streptavidin. (D) Jurkat cells (1×10 5 cells) were infected with the same amount (1 ng p24/200 µl) of E2 71 eMA or E2 71 mSAH pseudotypes pre-incubated with or without biotinylated anti-hTfR1 antibody or biotinylated Fab fragment of anti-hTfR1 antibody at 200 ng/ml. Transgene (EGFP) expression was analyzed by flow cytometry 3 days post-transduction. The averages and standard derivations of triplicate experiments are shown. (E) The titers (TU/40 µg p24/ml) of VSV-G pseudotype and the E2 71 eMA and mSAH pseudotypes with or without biotin α–hTfR1 Fab conjugation. The titers were calculated by triplicated transduction of Jurkat cells with EGFP-expressing vectors. The averages and standard derivations are shown. (F) Flow cytometric profiles of Jurkat cells (1×10 5 ) transduced with VSV-G, E2 71 eMA or mSAH pseudotypes (1 ng p24/200 µl) conjugated with the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody (10 ng) in the presence or absence of nevirapine (1 µM).
    Figure Legend Snippet: Site-specific biotinylation of the anti-hTfR1 antibody. (A) Schematic representation of conjugation of antibodies randomly biotinylated at lysine residues, the Fab fragment specifically biotinylated at C-terminal cysteines, and the biotinylated EGF. (B) ① disulfide bonds between heavy chains of anti-hTfR1 antibody are reduced by 2-mercaptethanolamine; ② the reduced cysteines were biotinylated with maleimide biotin; and ③ Fc regions of the biotinylated antibody are digested with pepsin. (C) ① Biotinylated anti-hTfR1 antibody; and ② the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody subjected to SDS-PAGE, followed by SyproRuby staining and western blotting, using HRP-conjugated streptavidin. (D) Jurkat cells (1×10 5 cells) were infected with the same amount (1 ng p24/200 µl) of E2 71 eMA or E2 71 mSAH pseudotypes pre-incubated with or without biotinylated anti-hTfR1 antibody or biotinylated Fab fragment of anti-hTfR1 antibody at 200 ng/ml. Transgene (EGFP) expression was analyzed by flow cytometry 3 days post-transduction. The averages and standard derivations of triplicate experiments are shown. (E) The titers (TU/40 µg p24/ml) of VSV-G pseudotype and the E2 71 eMA and mSAH pseudotypes with or without biotin α–hTfR1 Fab conjugation. The titers were calculated by triplicated transduction of Jurkat cells with EGFP-expressing vectors. The averages and standard derivations are shown. (F) Flow cytometric profiles of Jurkat cells (1×10 5 ) transduced with VSV-G, E2 71 eMA or mSAH pseudotypes (1 ng p24/200 µl) conjugated with the Fab fragment of cysteine-biotinylated anti-hTfR1 antibody (10 ng) in the presence or absence of nevirapine (1 µM).

    Techniques Used: Conjugation Assay, SDS Page, Staining, Western Blot, Infection, Incubation, Expressing, Flow Cytometry, Transduction

    46) Product Images from "Network analysis of UBE3A/E6AP-associated proteins provides connections to several distinct cellular processes"

    Article Title: Network analysis of UBE3A/E6AP-associated proteins provides connections to several distinct cellular processes

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.01.021

    UBE3A promotes the degradation of ASPP2 by the proteasome A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.
    Figure Legend Snippet: UBE3A promotes the degradation of ASPP2 by the proteasome A.UBE3A coimmunoprecipitates ASPP2. HEK 293T cells were transfected with the indicated vectors. 48 hours after transfection the HA-tagged proteins were immunoprecipitated with anti-HA agarose beads. Protein extracts and immunoprecipitates were analyzed by SDS-PAGE and Western blot using antibodies against HA-tag and Actin. HBH-ASPP2 carrying a biotinylation signal in the HBH tag was detected using streptavidin-HRP. HA-UBE3A: UBE3A isoform 1 C820A, catalytically inactive. B. Coexpression of UBE3A reduces ASPP2 protein levels. HEK 293T cells were transfected with the corresponding vectors. 48 hours post transfection the cells were harvested and the protein extracts were analyzed by SDS-PAGE and Western blot. Proteins were detected using anti HA, V5 and actin antibodies, and streptavidin HRP. Of note, UBE3A runs as a double band while its catalytically inactive form runs as a single band. - indicates that the cells were transfected with the corresponding empty vector. WT: wild type, CA: catalytically inactive form of UBE3A.

    Techniques Used: Transfection, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation

    47) Product Images from "Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints"

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116237

    The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.
    Figure Legend Snippet: The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

    Techniques Used: Modification, Expressing, Recombinant, SDS Page, Mutagenesis, Variant Assay, Labeling

    The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).
    Figure Legend Snippet: The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).

    Techniques Used: Clone Assay, Expressing, Construct, Sequencing, Transfection, SDS Page, Recombinant, Labeling

    48) Product Images from "IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production"

    Article Title: IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production

    Journal: Blood

    doi: 10.1182/blood-2009-07-230631

    Peritoneal macrophages from SHIP −/− mice produce IL-6 . (A) IL-6 production of freshly isolated and purified macrophages from bone marrow, spleen, lung, and peritoneum of WT and SHIP −/− mice, analyzed by ELISA after a 6-hour culture. Data are mean ± SE of 3 identical experiments. (B) Subpopulations of peritoneal cells were identified by flow cytometry as macrophages (Mac-1 + , F4/80 + ), monocytes (Mac-1 + , F4/80 − , Ly-6C hi ), myeloid dendritic cells (CD11c + , Ly-6C + ), and neutrophils (Mac-1 + , Gr-1 + , Ly-6C + ) and analyzed for intracellular IL-6. Shown is the total number of each population that expressed intracellular IL-6. Data are mean ± SE of 3 animals. (C) IL-6 histogram of peritoneal macrophages (defined as Mac-1 + , F4/80 + ) from WT (dashed line) or SHIP −/− (solid line) mice. Unstained cells are shown in gray. (D) Peritoneal macrophages identified with anti–Mac-1 and anti-F4/80 antibodies. (E) Total cells from 7 separate animals.
    Figure Legend Snippet: Peritoneal macrophages from SHIP −/− mice produce IL-6 . (A) IL-6 production of freshly isolated and purified macrophages from bone marrow, spleen, lung, and peritoneum of WT and SHIP −/− mice, analyzed by ELISA after a 6-hour culture. Data are mean ± SE of 3 identical experiments. (B) Subpopulations of peritoneal cells were identified by flow cytometry as macrophages (Mac-1 + , F4/80 + ), monocytes (Mac-1 + , F4/80 − , Ly-6C hi ), myeloid dendritic cells (CD11c + , Ly-6C + ), and neutrophils (Mac-1 + , Gr-1 + , Ly-6C + ) and analyzed for intracellular IL-6. Shown is the total number of each population that expressed intracellular IL-6. Data are mean ± SE of 3 animals. (C) IL-6 histogram of peritoneal macrophages (defined as Mac-1 + , F4/80 + ) from WT (dashed line) or SHIP −/− (solid line) mice. Unstained cells are shown in gray. (D) Peritoneal macrophages identified with anti–Mac-1 and anti-F4/80 antibodies. (E) Total cells from 7 separate animals.

    Techniques Used: Mouse Assay, Isolation, Purification, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    49) Product Images from "CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes"

    Article Title: CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes

    Journal: The Journal of Experimental Medicine

    doi:

    CD8αβ mediates raft association of TCR/CD3. T1.4 hybridomas (5 × 10 7 ) expressing no CD8 (A), CD8αβ (B), CD8αβ′ (C), or CD8αβ′′ (D) were photoaffinity labeled at 26°C with K d - 125”IASA”-YIPSAEK(ABA)I (0.5–1.5 × 10 8 cpm/7 ml). After UV irradiation the washed cells were lysed in 0.5% Brij58 and the lysates fractionated on sucrose density gradients. Fractions were collected from the top and immunoprecipitated with anti-TCRCβ mAb H57. The immunoprecipitates were analyzed by SDS-PAGE and PhosphorImaging.
    Figure Legend Snippet: CD8αβ mediates raft association of TCR/CD3. T1.4 hybridomas (5 × 10 7 ) expressing no CD8 (A), CD8αβ (B), CD8αβ′ (C), or CD8αβ′′ (D) were photoaffinity labeled at 26°C with K d - 125”IASA”-YIPSAEK(ABA)I (0.5–1.5 × 10 8 cpm/7 ml). After UV irradiation the washed cells were lysed in 0.5% Brij58 and the lysates fractionated on sucrose density gradients. Fractions were collected from the top and immunoprecipitated with anti-TCRCβ mAb H57. The immunoprecipitates were analyzed by SDS-PAGE and PhosphorImaging.

    Techniques Used: Expressing, Labeling, Irradiation, Immunoprecipitation, SDS Page

    Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.
    Figure Legend Snippet: Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Western Blot

    The CD8β tail mediates CD8 association with TCR/CD3. (A–C) T1.4 hybridomas with the indicated CD8 expression were lysed in 0.3% NP-40, and lysates immunoprecipitated with anti-TCR mAb H57 (A and B), anti-CD8α mAb 53.6.72 (C, left), or with anti-CD8β mAb H35–17 (C, right). The samples were analyzed by SDS-PAGE and Western blotting with anti-CD8α antiserum (A and C) or anti-CD3ε antiserum (B). (D) T1.4 T cell hybridomas expressing CD8αα or CD8αβ were incubated with monomeric K d -SYIPSAEK(ABA)I complexes (1.16 μM) for 2 h at 0–4°C. After UV irradiation the cells were lysed in 0.3% NP-40. Lysates were immunoprecipitated with anti-TCR mAb H57 or anti-CD8α mAb 53.6.72, as indicated and the immunoprecipitates analyzed by SDS-PAGE and Western blotting with anti-CD8α antiserum.
    Figure Legend Snippet: The CD8β tail mediates CD8 association with TCR/CD3. (A–C) T1.4 hybridomas with the indicated CD8 expression were lysed in 0.3% NP-40, and lysates immunoprecipitated with anti-TCR mAb H57 (A and B), anti-CD8α mAb 53.6.72 (C, left), or with anti-CD8β mAb H35–17 (C, right). The samples were analyzed by SDS-PAGE and Western blotting with anti-CD8α antiserum (A and C) or anti-CD3ε antiserum (B). (D) T1.4 T cell hybridomas expressing CD8αα or CD8αβ were incubated with monomeric K d -SYIPSAEK(ABA)I complexes (1.16 μM) for 2 h at 0–4°C. After UV irradiation the cells were lysed in 0.3% NP-40. Lysates were immunoprecipitated with anti-TCR mAb H57 or anti-CD8α mAb 53.6.72, as indicated and the immunoprecipitates analyzed by SDS-PAGE and Western blotting with anti-CD8α antiserum.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Western Blot, Incubation, Irradiation

    50) Product Images from "Vasoactive Intestinal Polypeptide-Immunoreactive Interneurons within Circuits of the Mouse Basolateral Amygdala"

    Article Title: Vasoactive Intestinal Polypeptide-Immunoreactive Interneurons within Circuits of the Mouse Basolateral Amygdala

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2063-17.2018

    VGluT1+ and VGluT2+ axon terminals form synapses with different caliber dendrites of VIP+ INs of the mouse BLA. A , Injection of an AAV2/6-CBA-FLEX-GFP vector into the BLA of VIP-IRES-Cre mice enabled the expression of the reporter protein GFP in VIP+ INs. B , GFP was detected throughout the somatodendritic domain as well as among axons of VIP+ INs. C , Asymmetric synapse (arrow) formed by an axon terminal containing VGluT1 (gold/silver reaction) with the dendrite of a VIP+ IN (HRP-DAB reaction). D , Asymmetric synapse (arrow) formed by an axon terminal containing VGluT2 (gold/silver reaction) with the dendrite of a VIP+ IN (HRP-DAB reaction). E , Whisker plots indicate the diameter of VIP+ IN dendrites targeted by VGluT1+ ( n = 56, sampled from 3 animals) or VGluT2+ ( n = 62, sampled from 3 animals) axon terminals. F , Frequency distributions of the diameter of VIP+ IN dendrites targeted by VGluT1+ or VGluT2+ axon terminals. Scale bars: A , 250 μm; B , 20 μm; C , D , 250 nm.
    Figure Legend Snippet: VGluT1+ and VGluT2+ axon terminals form synapses with different caliber dendrites of VIP+ INs of the mouse BLA. A , Injection of an AAV2/6-CBA-FLEX-GFP vector into the BLA of VIP-IRES-Cre mice enabled the expression of the reporter protein GFP in VIP+ INs. B , GFP was detected throughout the somatodendritic domain as well as among axons of VIP+ INs. C , Asymmetric synapse (arrow) formed by an axon terminal containing VGluT1 (gold/silver reaction) with the dendrite of a VIP+ IN (HRP-DAB reaction). D , Asymmetric synapse (arrow) formed by an axon terminal containing VGluT2 (gold/silver reaction) with the dendrite of a VIP+ IN (HRP-DAB reaction). E , Whisker plots indicate the diameter of VIP+ IN dendrites targeted by VGluT1+ ( n = 56, sampled from 3 animals) or VGluT2+ ( n = 62, sampled from 3 animals) axon terminals. F , Frequency distributions of the diameter of VIP+ IN dendrites targeted by VGluT1+ or VGluT2+ axon terminals. Scale bars: A , 250 μm; B , 20 μm; C , D , 250 nm.

    Techniques Used: Injection, Crocin Bleaching Assay, Plasmid Preparation, Mouse Assay, Expressing, Whisker Assay

    VIP immunoreactivity in the mouse BLA. A , Sparse somata and boutons of VIP+ INs can be observed throughout the BLA, whereas the lateral subdivision of the central amygdala (CeA) shows a dense plexus of VIP-immunolabeled fibers. B , C , Photomicrographs of typical bipolar VIP+ INs as revealed by HRP-DAB immunocytochemistry. D , Photomicrograph of a GFP-labeled dendrite of a VIP+ IN. To visualize the full dendritic domain of VIP+ INs, the viral vector AAV2/6-CBA-FLEX-GFP was injected into the BLA of VIP-IRES-cre mice. The presence in these mice of cre-recombinase only in VIP+ cells enabled the selective expression of the reporter protein GFP. Arrows indicate sporadic spines filled with GFP. E , F , EMs of a stubby spine arising from immunoperoxidase-labeled small VIP+ dendrites. The spine head faces an axon terminal labeled for VGluT1 (gold/silver particles). Astr, Amygdala-striatal transition zone; at, axon terminal; BA, basal nucleus of the amygdala; CeA, central nucleus of the amygdala; ic, internal capsule; LA, lateral nucleus of the amygdala. Scale bars: A , 250 μm; B , C , 20 μm; D , 5 μm; E , 250 nm; F , 200 nm.
    Figure Legend Snippet: VIP immunoreactivity in the mouse BLA. A , Sparse somata and boutons of VIP+ INs can be observed throughout the BLA, whereas the lateral subdivision of the central amygdala (CeA) shows a dense plexus of VIP-immunolabeled fibers. B , C , Photomicrographs of typical bipolar VIP+ INs as revealed by HRP-DAB immunocytochemistry. D , Photomicrograph of a GFP-labeled dendrite of a VIP+ IN. To visualize the full dendritic domain of VIP+ INs, the viral vector AAV2/6-CBA-FLEX-GFP was injected into the BLA of VIP-IRES-cre mice. The presence in these mice of cre-recombinase only in VIP+ cells enabled the selective expression of the reporter protein GFP. Arrows indicate sporadic spines filled with GFP. E , F , EMs of a stubby spine arising from immunoperoxidase-labeled small VIP+ dendrites. The spine head faces an axon terminal labeled for VGluT1 (gold/silver particles). Astr, Amygdala-striatal transition zone; at, axon terminal; BA, basal nucleus of the amygdala; CeA, central nucleus of the amygdala; ic, internal capsule; LA, lateral nucleus of the amygdala. Scale bars: A , 250 μm; B , C , 20 μm; D , 5 μm; E , 250 nm; F , 200 nm.

    Techniques Used: Immunolabeling, Immunocytochemistry, Labeling, Plasmid Preparation, Crocin Bleaching Assay, Injection, Mouse Assay, Expressing

    51) Product Images from "PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity"

    Article Title: PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028611

    PEGylated TNYL-RAW inhibits capillary-like tube formation in co-cultured HUVECs and EMCs. EMCs expressing EGFP and cultured in DMEM or complete Medium 200 and HUVECs labeled with CellTracker™ Orange were plated on Matrigel individually or mixed at a 1∶2 ratio and imaged 5 and 20 hours later. The effect of 5 µM PEG-TNYL-RAW or an equal amount of PEG control on tube formation was analyzed. The histogram shows average tube lengths quantified from 2–3 micrographs at 5 hours for EMCs and 20 hours for HUVECs and HUVEC + EMC co-cultures and normalized to the average for the PEG control. Error bars represent the standard error from tube length measured from 3–4 wells. Tube length in the presence of PEG-TNYL-RAW was compared to that in the presence of PEG by one-way ANOVA and Bonferroni's post test. ***P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits capillary-like tube formation in co-cultured HUVECs and EMCs. EMCs expressing EGFP and cultured in DMEM or complete Medium 200 and HUVECs labeled with CellTracker™ Orange were plated on Matrigel individually or mixed at a 1∶2 ratio and imaged 5 and 20 hours later. The effect of 5 µM PEG-TNYL-RAW or an equal amount of PEG control on tube formation was analyzed. The histogram shows average tube lengths quantified from 2–3 micrographs at 5 hours for EMCs and 20 hours for HUVECs and HUVEC + EMC co-cultures and normalized to the average for the PEG control. Error bars represent the standard error from tube length measured from 3–4 wells. Tube length in the presence of PEG-TNYL-RAW was compared to that in the presence of PEG by one-way ANOVA and Bonferroni's post test. ***P

    Techniques Used: Cell Culture, Expressing, Labeling

    Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.
    Figure Legend Snippet: Modified forms of TNYL-RAW retain high EphB4 binding affinity and potency for inhibition of EphB4-ephrin-B2 binding. (A) Biotinylated, streptavidin-bound and PEGylated TNYL-RAW were incubated at the indicated concentrations in EphB4-coated ELISA wells. Biotinylated TNYL-RAW was detected with streptavidin-HRP, TNYL-RAW-streptavidin was detected with and anti-streptavidin antibody coupled to HRP, and PEG-TNYL-RAW was detected with an anti-PEG antibody followed by a secondary antibody conjugated to HRP. (B) The indicated concentrations of EphB4 AP were incubated in ELISA wells pre-coated with streptavidin and biotinylated TNYL-RAW (left) or an anti-IgG antibody and TNYL-RAW-Fc (right). K d values are based on EphB4 AP concentrations calculated from AP activity. (C) The different forms of TNYL-RAW were incubated at the indicated concentrations together with a constant amount of ephrin-B2 AP in ELISA wells pre-coated with EphB4 Fc. The ratio of ephrin-B2 AP bound in the presence and in the absence of peptide is shown. The graphs show averages ± SE from triplicate measurements in representative experiments, while the K d and IC 50 values are calculated from 3 to 11 experiments.

    Techniques Used: Modification, Binding Assay, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay

    The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.
    Figure Legend Snippet: The TNYL-RAW peptide is rapidly lost in cell culture medium and from the mouse circulation. (A) Biotinylated TNYL-RAW peptide was incubated with cultured PC3 prostate cancer cells grown in the same medium for 3 days or in the culture medium freshly replaced just before adding the peptide. Functional (EphB4- and streptavidin-binding) peptide remaining at the indicated times was captured in ELISA plates coated with EphB4 Fc and detected with streptavidin-HRP. (B) TNYL-RAW was incubated at 37°C in PC3 cell conditioned medium (without cells) with and without a mixture of protease inhibitors including aprotinin, leupeptin, pepstatin and PMSF, and detected as in (A). (C) TNYL-RAW was incubated with PC3 cell conditioned medium for 4 hours and added together with ephrin-B2 AP to ELISA wells pre-coated with EphB4 Fc. TNYL-RAW mixed with conditioned medium right before the ELISA assay (0 hrs) was used as a control. The graph shows the ratio of ephrin-B2 AP bound in the presence and in the absence of peptide. (D) Serum from 3 mice injected intravenously with 6 nmoles biotinylated TNYL-RAW was collected 30 min after peptide administration and incubated at a dilution of 1∶20 in ELISA wells pre-coated with EphB4 Fc. Based on the amount of injected TNYL-RAW and an estimated mouse serum volume of 2.5 ml, the peptide concentration in the wells would be 120 nM. TNYL-RAW at a concentration of 5 nM in similarly diluted mouse serum was used for comparison. Bound peptide was detected with streptavidin-HRP. (E) TNYL-RAW was incubated in undiluted mouse serum ex vivo for the indicated times and detected as described in (A). Averages from 3 measurements ± SE are shown in all the panels.

    Techniques Used: Cell Culture, Incubation, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Concentration Assay, Ex Vivo

    PEGylation increases the effectiveness of the TNYL-RAW peptide in inhibiting capillary-like tube formation by co-coltured HUVECs and EMCs. HUVECs labeled with CellTracker™ Orange and EMCs expressing EGFP were plated on Matrigel in complete Medium 200 at a 2∶1 ratio and imaged 15 hours later. The effect of different concentrations of PEG-TNYL-RAW or TNYL-RAW on tube formation was analyzed. The histograms show the average tube lengths for the different peptide treatments normalized to the average for the PEG or DMSO controls. Error bars represent the standard error from 3–4 wells. Tube lengths for PEG-TNYL-RAW or TNYL-RAW were compared to the PEG or DMSO control by one-way ANOVA and Dunnett's post test. **P
    Figure Legend Snippet: PEGylation increases the effectiveness of the TNYL-RAW peptide in inhibiting capillary-like tube formation by co-coltured HUVECs and EMCs. HUVECs labeled with CellTracker™ Orange and EMCs expressing EGFP were plated on Matrigel in complete Medium 200 at a 2∶1 ratio and imaged 15 hours later. The effect of different concentrations of PEG-TNYL-RAW or TNYL-RAW on tube formation was analyzed. The histograms show the average tube lengths for the different peptide treatments normalized to the average for the PEG or DMSO controls. Error bars represent the standard error from 3–4 wells. Tube lengths for PEG-TNYL-RAW or TNYL-RAW were compared to the PEG or DMSO control by one-way ANOVA and Dunnett's post test. **P

    Techniques Used: Labeling, Expressing

    PEGylated TNYL-RAW inhibits capillary sprouting in co-coltured HUVECs and EMCs. Collagen embedded spheroids generated with HUVECs expressing mCherry, EMCs expressing EGFP or a 1∶1 mixture of the two cell types were treated with 5 µM PEG-TNYL-RAW or PEG for 2 days. The number of sprouts and the cumulative sprout length in the HUVE + EMC spheroids were normalized to the average for the PEG control. The histogram shows averages from 40–45 spheroids ± SE. The values obtained for spheroids treated with PEG-TNYL-RAW were compared to those with PEG control by one-way ANOVA. ***P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits capillary sprouting in co-coltured HUVECs and EMCs. Collagen embedded spheroids generated with HUVECs expressing mCherry, EMCs expressing EGFP or a 1∶1 mixture of the two cell types were treated with 5 µM PEG-TNYL-RAW or PEG for 2 days. The number of sprouts and the cumulative sprout length in the HUVE + EMC spheroids were normalized to the average for the PEG control. The histogram shows averages from 40–45 spheroids ± SE. The values obtained for spheroids treated with PEG-TNYL-RAW were compared to those with PEG control by one-way ANOVA. ***P

    Techniques Used: Generated, Expressing

    Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P
    Figure Legend Snippet: Modified forms of TNYL-RAW have increased stability in cell culture medium and in the mouse circulation. (A, B) Biotinylated, streptavidin-bound, fused to Fc and PEGylated TNYL-RAW were incubated in medium conditioned by PC3 prostate cancer cells (A) or mouse serum (B). Functional peptide remaining at the indicated times was captured in ELISA plates and quantified. Biotinylated TNYL-RAW was captured on ELISA wells pre-coated with EphB4 Fc and detected with Streptavidin-HRP. TNYL-RAW-streptavidin was captured on wells pre-coated with EphB4 Fc and detected with an anti-streptavidin antibody coupled to HRP. TNYL-RAW-Fc was captured on wells coated with an anti-Fc antibody and detected with EphB4 AP. PEG-TNYL-RAW was captured on wells coated with EphB4 Fc and detected with anti-PEG antibody followed by a secondary antibody conjugated to HRP. Normalized averages from 6–9 measurements ± SE are shown. Peptide amounts at different time points were compared to those at time 0 by one-way ANOVA and Dunnett's post test. *P

    Techniques Used: Modification, Cell Culture, Incubation, Functional Assay, Enzyme-linked Immunosorbent Assay

    PEGylated TNYL-RAW inhibits tyrosine phosphorylation of EphB4 and ephrin-B2. (A) B16 melanoma cells pretreated with the indicated concentrations of PEG-TNYL-RAW or TNYL-RAW for 15 min or 24 hours were stimulated with 1.5 µg/ml preclustered ephrin-B2 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the peptide. EphB4 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (B) The inhibition curve shows the relative levels of EphB4 phosphorylation in the presence of different concentrations of PEG-TNYL-RAW, which were quantified from immunoblots and normalized to the amount of immunoprecipitated EphB4. Error bars represent the standard error from 3–6 experiments. (C) HUVEC and EMC lysates were probed for EphB4, ephrin-B2 (band at ∼45 Kd detected with a pan-ephrin-B antibody) and ß-actin as a loading control. It is not known why the ephrin-B2 band appears as a more prominent doublet in EMCs than HUVECs. (D) HUVECs and EMCs were cultured individually or mixed at a 1∶1 ratio in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. EphB4 immunoprecipitates were probed with an anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (E) HUVECs and EMCs, which express EGFP, were cultured at a 1∶1 ratio for 15 hours in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. The cells were then stimulated with 1.5 µg/ml preclustered EphB4 Fc or Fc as a control for 20 min in the continued presence of the peptide or PEG. The cells were stained for phospho-ephrin-B (red), which likely corresponds to the phosphorylated form of the EphB4 preferred ligand ephrin-B2, and nuclei were labeled with DAPI (blue). Scale bar = 50 µM. Fluorescence intensity from 6 micrographs per condition was quantified. The values obtained (expressed in arbitrary units ± standard error) are: PEG Fc, 24±2.6; PEG EphB4 Fc, 44±3.6; PEG-TNYL-RAW EphB4 Fc, 20±2.5. The fluorescence of cells treated with PEG-TNYL-RAW EphB4 Fc was significantly (P
    Figure Legend Snippet: PEGylated TNYL-RAW inhibits tyrosine phosphorylation of EphB4 and ephrin-B2. (A) B16 melanoma cells pretreated with the indicated concentrations of PEG-TNYL-RAW or TNYL-RAW for 15 min or 24 hours were stimulated with 1.5 µg/ml preclustered ephrin-B2 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the peptide. EphB4 immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (B) The inhibition curve shows the relative levels of EphB4 phosphorylation in the presence of different concentrations of PEG-TNYL-RAW, which were quantified from immunoblots and normalized to the amount of immunoprecipitated EphB4. Error bars represent the standard error from 3–6 experiments. (C) HUVEC and EMC lysates were probed for EphB4, ephrin-B2 (band at ∼45 Kd detected with a pan-ephrin-B antibody) and ß-actin as a loading control. It is not known why the ephrin-B2 band appears as a more prominent doublet in EMCs than HUVECs. (D) HUVECs and EMCs were cultured individually or mixed at a 1∶1 ratio in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. EphB4 immunoprecipitates were probed with an anti-phosphotyrosine antibody (PTyr) and reprobed for EphB4. (E) HUVECs and EMCs, which express EGFP, were cultured at a 1∶1 ratio for 15 hours in the presence of 1.5 µM PEG-TNYL-RAW or PEG control. The cells were then stimulated with 1.5 µg/ml preclustered EphB4 Fc or Fc as a control for 20 min in the continued presence of the peptide or PEG. The cells were stained for phospho-ephrin-B (red), which likely corresponds to the phosphorylated form of the EphB4 preferred ligand ephrin-B2, and nuclei were labeled with DAPI (blue). Scale bar = 50 µM. Fluorescence intensity from 6 micrographs per condition was quantified. The values obtained (expressed in arbitrary units ± standard error) are: PEG Fc, 24±2.6; PEG EphB4 Fc, 44±3.6; PEG-TNYL-RAW EphB4 Fc, 20±2.5. The fluorescence of cells treated with PEG-TNYL-RAW EphB4 Fc was significantly (P

    Techniques Used: Inhibition, Western Blot, Immunoprecipitation, Cell Culture, Staining, Labeling, Fluorescence

    52) Product Images from "Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder"

    Article Title: Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder

    Journal: Scientific Reports

    doi: 10.1038/srep36110

    Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p
    Figure Legend Snippet: Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p

    Techniques Used: Expressing

    Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p
    Figure Legend Snippet: Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p

    Techniques Used: Staining, TUNEL Assay

    53) Product Images from "Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation"

    Article Title: Screening of Proximal and Interacting Proteins in Rice Protoplasts by Proximity-Dependent Biotinylation

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.00749

    BirAG can biotinylate itself and proteins in rice protoplasts. (A) Expression and localization of BirA ∗ and BirAG proteins in rice protoplasts. Plasmids used for protoplasts transfection were indicated. (B) Western-blot of biotinylated proteins in rice protoplasts after transient expression of BirA-FLAG, BirA ∗ -FLAG, and BirAG-FLAG, with or without biotin at 50 μM in protoplasts incubation solution. Different plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and different BirA versions, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. p35S, CaMV35S promoter; pUbi, maize ubiquitin promoter; arrow indicate the biotinylated BirAG proteins.
    Figure Legend Snippet: BirAG can biotinylate itself and proteins in rice protoplasts. (A) Expression and localization of BirA ∗ and BirAG proteins in rice protoplasts. Plasmids used for protoplasts transfection were indicated. (B) Western-blot of biotinylated proteins in rice protoplasts after transient expression of BirA-FLAG, BirA ∗ -FLAG, and BirAG-FLAG, with or without biotin at 50 μM in protoplasts incubation solution. Different plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and different BirA versions, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. p35S, CaMV35S promoter; pUbi, maize ubiquitin promoter; arrow indicate the biotinylated BirAG proteins.

    Techniques Used: Expressing, Transfection, Western Blot, Incubation, Staining

    Factors influencing BirAG on biotinylating proteins in rice protoplasts. (A) Biotinylation of proteins increases with the biotin concentration in protoplasts incubation buffer. (B) Influence of ATP on protein biotinylation in rice protoplasts, ATP was added into the protoplasts incubation buffer at the concentration of 10 μM. (C) Influence of protoplasts incubation time and biotin concentrations on protein biotinylation in rice protoplasts. (D) BirAG fused with different target proteins can biotinylated proteins in rice protoplasts. Different treatment and plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and BirAG, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. pUbi, maize ubiquitin promoter; arrows indicate the biotinylated BirAG proteins.
    Figure Legend Snippet: Factors influencing BirAG on biotinylating proteins in rice protoplasts. (A) Biotinylation of proteins increases with the biotin concentration in protoplasts incubation buffer. (B) Influence of ATP on protein biotinylation in rice protoplasts, ATP was added into the protoplasts incubation buffer at the concentration of 10 μM. (C) Influence of protoplasts incubation time and biotin concentrations on protein biotinylation in rice protoplasts. (D) BirAG fused with different target proteins can biotinylated proteins in rice protoplasts. Different treatment and plasmids used for protoplasts transfection were indicated. Streptavidin-HRP and Anti-FLAG antibody were used for detection of biotinylated proteins and BirAG, respectively. Coomassie Brilliant Blue (CBB G-250) staining was used for monitoring the sample loading amount. pUbi, maize ubiquitin promoter; arrows indicate the biotinylated BirAG proteins.

    Techniques Used: Concentration Assay, Incubation, Transfection, Staining

    54) Product Images from "Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds *"

    Article Title: Identification of Bacterial Target Proteins for the Salicylidene Acylhydrazide Class of Virulence-blocking Compounds *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.233858

    Far Western probing to examine binding of ME0052-Bio to putative target proteins. A , putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx ( 1 ), FolX ( 2 ), z2714 ( 3 ), z3974 ( 4 ), WrbA ( 5 ), SurA ( 6 ), and StcE ( 7 ). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 ( EC ), Y. pseudotuberculosis ( YP ), S. typhimurium ( ST ), P. aeruginosa ( PA ), and S. flexneri ( SF ) or empty vector control (− ve ). Panels show overexpression of target proteins: WrbA ( B ), Tpx ( C ), FolX and FolB ( D ). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio ( E ). The addition of 200 μ m unlabeled ME0052 ( F–H ) strongly affected the binding of the biotinylated probe.
    Figure Legend Snippet: Far Western probing to examine binding of ME0052-Bio to putative target proteins. A , putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx ( 1 ), FolX ( 2 ), z2714 ( 3 ), z3974 ( 4 ), WrbA ( 5 ), SurA ( 6 ), and StcE ( 7 ). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 ( EC ), Y. pseudotuberculosis ( YP ), S. typhimurium ( ST ), P. aeruginosa ( PA ), and S. flexneri ( SF ) or empty vector control (− ve ). Panels show overexpression of target proteins: WrbA ( B ), Tpx ( C ), FolX and FolB ( D ). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio ( E ). The addition of 200 μ m unlabeled ME0052 ( F–H ) strongly affected the binding of the biotinylated probe.

    Techniques Used: Western Blot, Binding Assay, Clone Assay, Lysis, Negative Control, Plasmid Preparation, Over Expression, Mutagenesis

    55) Product Images from "Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *"

    Article Title: Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.622274

    FACS analysis of v5-tagged MMP-9 variant recruitment to living MRC-5 cells. A , live MRC-5 cells incubated with v5-tagged MMP-9 were stained with mouse anti-v5 or an irrelevant mouse isotype-matched antibody followed by anti-mouse Alexa Fluor 488-conjugated antibody. Single cells (69.9) were gated after doublet exclusion, and DAPI-negative cells (42.5) were considered to be live. The shift in the histogram between isotype-matched (0.7) and anti-v5 antibody (29.0) profiles shows MMP-9 presence on the MRC-5 cell surface. B , live MRC-5 cells incubated with v5-tagged ΔFN were stained with anti-v5 or an irrelevant mouse isotype-matched antibody followed by Alexa Fluor 488-conjugated anti-mouse antibody. No shift between isotype-matched (0.83) and anti-v5 antibody peaks (5.89) is observed. C , superposition of isotype-matched ( orange ) and anti-v5 antibody (ΔFN, blue ; MMP-9, green ) staining profiles. SS-A , side scatter area; FS-A , forward scatter area; FS-W , forward scatter width.
    Figure Legend Snippet: FACS analysis of v5-tagged MMP-9 variant recruitment to living MRC-5 cells. A , live MRC-5 cells incubated with v5-tagged MMP-9 were stained with mouse anti-v5 or an irrelevant mouse isotype-matched antibody followed by anti-mouse Alexa Fluor 488-conjugated antibody. Single cells (69.9) were gated after doublet exclusion, and DAPI-negative cells (42.5) were considered to be live. The shift in the histogram between isotype-matched (0.7) and anti-v5 antibody (29.0) profiles shows MMP-9 presence on the MRC-5 cell surface. B , live MRC-5 cells incubated with v5-tagged ΔFN were stained with anti-v5 or an irrelevant mouse isotype-matched antibody followed by Alexa Fluor 488-conjugated anti-mouse antibody. No shift between isotype-matched (0.83) and anti-v5 antibody peaks (5.89) is observed. C , superposition of isotype-matched ( orange ) and anti-v5 antibody (ΔFN, blue ; MMP-9, green ) staining profiles. SS-A , side scatter area; FS-A , forward scatter area; FS-W , forward scatter width.

    Techniques Used: FACS, Variant Assay, Incubation, Staining

    56) Product Images from "Role of T cell Transforming Growth Factor ? Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection 1"

    Article Title: Role of T cell Transforming Growth Factor ? Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection 1

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

    doi: 10.4049/jimmunol.0902446

    IgG, C3d and C4d deposition in allografts of WT and CD4-DNR recipients WT (left column) or CD4-DNR (right column) allograft recipients were treated with inductive anti-CD4 mAb therapy. Grafts were recovered at either the time of rejection or 40 days after transplantation. Graft sections were fixed and incubated with goat anti-mouse IgG (A) or the goat anti-mouse C3d or C4d (B) followed by development with 3-amino-9-ethylcarbazole or DAB to visualize mouse Ab and complement deposition. Results are representative of grafts from 6-10 recipients. Magnification, 400× (A) and 200× (B) .
    Figure Legend Snippet: IgG, C3d and C4d deposition in allografts of WT and CD4-DNR recipients WT (left column) or CD4-DNR (right column) allograft recipients were treated with inductive anti-CD4 mAb therapy. Grafts were recovered at either the time of rejection or 40 days after transplantation. Graft sections were fixed and incubated with goat anti-mouse IgG (A) or the goat anti-mouse C3d or C4d (B) followed by development with 3-amino-9-ethylcarbazole or DAB to visualize mouse Ab and complement deposition. Results are representative of grafts from 6-10 recipients. Magnification, 400× (A) and 200× (B) .

    Techniques Used: Transplantation Assay, Incubation

    57) Product Images from "Iba-1-/CD68+ microglia are a prominent feature of age-associated deep subcortical white matter lesions"

    Article Title: Iba-1-/CD68+ microglia are a prominent feature of age-associated deep subcortical white matter lesions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210888

    Quantification of microglial cell expression. The immunoreactive mean area of Iba-1 (A), CD68 (B) and MHCII (C) across the cohort. *P
    Figure Legend Snippet: Quantification of microglial cell expression. The immunoreactive mean area of Iba-1 (A), CD68 (B) and MHCII (C) across the cohort. *P

    Techniques Used: Expressing

    Microglial immunohistochemistry. Distinct microglial cell populations are evident within the deep subcortical lesion (DSCL). CD68 + amoeboid microglial (red) were indicated by the black arrow and separate more ramified cells were labelled with Iba-1 (brown) indicated by the blue arrow. Dual staining showed some colocalisation of CD68 (red) and Iba-1 (brown) indicated by the red arrow. Scale bar = 50μm.
    Figure Legend Snippet: Microglial immunohistochemistry. Distinct microglial cell populations are evident within the deep subcortical lesion (DSCL). CD68 + amoeboid microglial (red) were indicated by the black arrow and separate more ramified cells were labelled with Iba-1 (brown) indicated by the blue arrow. Dual staining showed some colocalisation of CD68 (red) and Iba-1 (brown) indicated by the red arrow. Scale bar = 50μm.

    Techniques Used: Immunohistochemistry, Staining

    Microglial morphology analysis. Iba-1 labelled microglial increase in size and roundness (M-score) throughout the three groups, indicative of an increasing amoeboid, phagocytic phenotype between DSCL vs NAWM and NAWM vs control cases (A). CD68-labelled microglia also increase in size and roundness, indicative of amoeboid, phagocytic phenotype between DSCL vs NAWM and control cases (B). MHCII-labelled microglia show no significant difference in their M-score between the three groups (C). *P
    Figure Legend Snippet: Microglial morphology analysis. Iba-1 labelled microglial increase in size and roundness (M-score) throughout the three groups, indicative of an increasing amoeboid, phagocytic phenotype between DSCL vs NAWM and NAWM vs control cases (A). CD68-labelled microglia also increase in size and roundness, indicative of amoeboid, phagocytic phenotype between DSCL vs NAWM and control cases (B). MHCII-labelled microglia show no significant difference in their M-score between the three groups (C). *P

    Techniques Used:

    Microglial immunostaining. Iba-1 (A-C), CD68 (D-F) and MHCII (G-I) protein expression within the control, NAWM and DSCL cases. Control WM showed a regular distribution of Iba-1 positive ramified microglia (A) in contrast to the little CD68 and MHCII immunostaining in control WM (D G). Within NAWM an increase in immunoreactivity was evident across all three microglial markers (B,E,H). Within DSCL, Iba-1 and MHCII immunostaining highlighted both ramified and amoeboid microglia (C I). In contrast CD68 immunostaining highlighted amoeboid microglia (F). Scale bar = 100μm. NAWM: normal appearing white matter, DSCL: deep subcortical lesion, WM: white matter, MHCII: major histocompatibility complex II.
    Figure Legend Snippet: Microglial immunostaining. Iba-1 (A-C), CD68 (D-F) and MHCII (G-I) protein expression within the control, NAWM and DSCL cases. Control WM showed a regular distribution of Iba-1 positive ramified microglia (A) in contrast to the little CD68 and MHCII immunostaining in control WM (D G). Within NAWM an increase in immunoreactivity was evident across all three microglial markers (B,E,H). Within DSCL, Iba-1 and MHCII immunostaining highlighted both ramified and amoeboid microglia (C I). In contrast CD68 immunostaining highlighted amoeboid microglia (F). Scale bar = 100μm. NAWM: normal appearing white matter, DSCL: deep subcortical lesion, WM: white matter, MHCII: major histocompatibility complex II.

    Techniques Used: Immunostaining, Expressing

    58) Product Images from "CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes"

    Article Title: CD8? Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56lck Complexes

    Journal: The Journal of Experimental Medicine

    doi:

    Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.
    Figure Legend Snippet: Palmitoylation of CD8β is essential for CD8 localization in rafts and efficient lck association. (A) T1.4 hybridomas with the indicated CD8 expression were surface-biotinylated, lysed in cold TX-100 (1%), and fractionated in M and DIM fractions. These were immunoprecipitated with anti-CD8α mAb 53.6.72. The samples were analyzed by SDS-PAGE and Western blotting with streptavidin. (B–E) T1.4 hybridomas with the indicated CD8 expression were lysed in Brij96 (1%) and the lysates immunoprecipitated with mAb 53.6.72 (B and D) or anti-CD8β mAB H35–17 (C and E). The immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-lck mAb 3A5 (B and C) or anti-CD8α antiserum (D and E). Note that anti-CD8β mAb H35 is unable to precipitate CD8αα and CD8αα β . One out of two experiments is shown.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Western Blot

    59) Product Images from "IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses"

    Article Title: IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20091738

    IL-21–deficient mice form Tfh cells after immunization, but their maintenance is impaired. (A) Flow cytometric contour plots and graphical analysis of CXCR5 + PD-1 + Tfh cells gated on CD4 + B220 − live lymphocytes from Il21 +/+ and Il21 −/− mice at the indicated time points after SRBC immunization (percentages are shown). (B) Photomicrographs of spleen sections taken from Il21 +/+ (top) and Il21 −/− (bottom) mice 8 d after immunization with SRBCs. In all panels, IgD is stained in brown; blue color stains indicate PNA binding (left), CD3 (middle), and PD-1 (right). Bars, 200 µm. Statistically significant differences are indicated (*, P ≤ 0.05). Data are representative of two independent experiments, each symbol represents one mouse, and tops of bars are drawn through the median values. ns, not significant.
    Figure Legend Snippet: IL-21–deficient mice form Tfh cells after immunization, but their maintenance is impaired. (A) Flow cytometric contour plots and graphical analysis of CXCR5 + PD-1 + Tfh cells gated on CD4 + B220 − live lymphocytes from Il21 +/+ and Il21 −/− mice at the indicated time points after SRBC immunization (percentages are shown). (B) Photomicrographs of spleen sections taken from Il21 +/+ (top) and Il21 −/− (bottom) mice 8 d after immunization with SRBCs. In all panels, IgD is stained in brown; blue color stains indicate PNA binding (left), CD3 (middle), and PD-1 (right). Bars, 200 µm. Statistically significant differences are indicated (*, P ≤ 0.05). Data are representative of two independent experiments, each symbol represents one mouse, and tops of bars are drawn through the median values. ns, not significant.

    Techniques Used: Mouse Assay, Flow Cytometry, Staining, Binding Assay

    IL-21R expression is required on both GC B cells and Tfh cells for their maintenance but is dispensable for their formation. (A and B) Gating strategy (A) and bar graphs (B) of mixed bone marrow chimeras containing a 1:1 ratio of control Il21r +/+ CD45.1/ Il21r +/+ CD45.2 or Il21r +/+ CD45.1/ Il21r −/− CD45.2 bone marrow. Spleens from unimmunized mice (A; and B, top) or from mice immunized with SRBCs 6 or 14 d previously (B, middle and bottom) were analyzed by flow cytometry for the percentage of B220 + B cells that are CD45.2 + (B220 + ), the percentage of GL-7 + Fas + GC cells among B220 + cells that are CD45.2 + (GC cells), the percentage of CD4 + Th cells that are CD45.2 + (CD4 + ), and the percentage of CXCR5 high PD-1 high Tfh cells among CD4 + cells that are CD45.2 + (Tfh cells). Each bar represents a single recipient mouse; individual mice have been numbered and placed in the same order in each of the plots. Statistically significant decreases are indicated (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Data are representative of two independent experiments. (C and D) Gating strategy (C) and dot plots (D) of the proportion of B220 + , GC (B220 + Fas + GL-7 + ), and IgG1 + GC (B220 + Fas + GL-7 + IgG1 + ) cells derived from the CD45.2 compartment of mixed chimeras reconstituted with a 1:1 ratio of either CD45.1 Il21r +/+ /CD45.2 Il21r +/+ (left) or CD45.1 Il21r +/+ /CD45.2 Il21r −/− (right) bone marrow before (C; and D, top) or 6 d after SRBC immunization (D, bottom). In D, each number represents a single recipient mouse from two separate groups of the chimeric mice indicated above the left and right panels.
    Figure Legend Snippet: IL-21R expression is required on both GC B cells and Tfh cells for their maintenance but is dispensable for their formation. (A and B) Gating strategy (A) and bar graphs (B) of mixed bone marrow chimeras containing a 1:1 ratio of control Il21r +/+ CD45.1/ Il21r +/+ CD45.2 or Il21r +/+ CD45.1/ Il21r −/− CD45.2 bone marrow. Spleens from unimmunized mice (A; and B, top) or from mice immunized with SRBCs 6 or 14 d previously (B, middle and bottom) were analyzed by flow cytometry for the percentage of B220 + B cells that are CD45.2 + (B220 + ), the percentage of GL-7 + Fas + GC cells among B220 + cells that are CD45.2 + (GC cells), the percentage of CD4 + Th cells that are CD45.2 + (CD4 + ), and the percentage of CXCR5 high PD-1 high Tfh cells among CD4 + cells that are CD45.2 + (Tfh cells). Each bar represents a single recipient mouse; individual mice have been numbered and placed in the same order in each of the plots. Statistically significant decreases are indicated (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Data are representative of two independent experiments. (C and D) Gating strategy (C) and dot plots (D) of the proportion of B220 + , GC (B220 + Fas + GL-7 + ), and IgG1 + GC (B220 + Fas + GL-7 + IgG1 + ) cells derived from the CD45.2 compartment of mixed chimeras reconstituted with a 1:1 ratio of either CD45.1 Il21r +/+ /CD45.2 Il21r +/+ (left) or CD45.1 Il21r +/+ /CD45.2 Il21r −/− (right) bone marrow before (C; and D, top) or 6 d after SRBC immunization (D, bottom). In D, each number represents a single recipient mouse from two separate groups of the chimeric mice indicated above the left and right panels.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Derivative Assay

    IL-21 contributes to affinity maturation but is dispensable for EM B cell formation. (A) Representative flow cytometric contour plots of donor CD45.1 SW HEL cells from the spleens of Il21 +/+ or Il21 −/− recipient mice 10 d after adoptive transfer and immunization with HEL 3X SRBCs. Contour plots show IgG1 + cells binding with high (top oval gates) and low (bottom oval gates) affinity to HEL 3X (percentages are shown). (B) Bar graphs show HEL 3X hi IgG1 + CD45.1 cells (top oval gates in A) as a proportion of all HEL 3X -binding IgG1 + cells (square gates in A). This figure is representative of two independent experiments ( n ≥ 3 mice per group. (C) Ratio of total Tfh (CXCR5 high PD-1 high CD4 + ) cells to total HEL-binding donor (CD45.1) GC B cells (Fas + GL-7 + ). Two independent experiments are represented in this plot. (D) Representative flow cytometric contour plots 10 d after adoptive transfer of SW HEL B cells into Il21 +/+ or Il21 −/− mice and immunization with HEL 3X SRBCs gated on donor CD45.1 and HEL-binding cells (percentages are shown). The gates show GC cells (HEL int binding) and EM B cells (HEL hi binding). (E) Phenotype of GC B cells and EM cells gated as in D. (F and G) Total number of donor HEL int -binding GC B cells per spleen (F) and the proportion of donor HEL hi -binding EM B cells (G) in mice of the indicated genotypes gated as in D. Each symbol represents one mouse and tops of bars are drawn through the median values. Statistically significant differences are indicated (**, P ≤ 0.01).
    Figure Legend Snippet: IL-21 contributes to affinity maturation but is dispensable for EM B cell formation. (A) Representative flow cytometric contour plots of donor CD45.1 SW HEL cells from the spleens of Il21 +/+ or Il21 −/− recipient mice 10 d after adoptive transfer and immunization with HEL 3X SRBCs. Contour plots show IgG1 + cells binding with high (top oval gates) and low (bottom oval gates) affinity to HEL 3X (percentages are shown). (B) Bar graphs show HEL 3X hi IgG1 + CD45.1 cells (top oval gates in A) as a proportion of all HEL 3X -binding IgG1 + cells (square gates in A). This figure is representative of two independent experiments ( n ≥ 3 mice per group. (C) Ratio of total Tfh (CXCR5 high PD-1 high CD4 + ) cells to total HEL-binding donor (CD45.1) GC B cells (Fas + GL-7 + ). Two independent experiments are represented in this plot. (D) Representative flow cytometric contour plots 10 d after adoptive transfer of SW HEL B cells into Il21 +/+ or Il21 −/− mice and immunization with HEL 3X SRBCs gated on donor CD45.1 and HEL-binding cells (percentages are shown). The gates show GC cells (HEL int binding) and EM B cells (HEL hi binding). (E) Phenotype of GC B cells and EM cells gated as in D. (F and G) Total number of donor HEL int -binding GC B cells per spleen (F) and the proportion of donor HEL hi -binding EM B cells (G) in mice of the indicated genotypes gated as in D. Each symbol represents one mouse and tops of bars are drawn through the median values. Statistically significant differences are indicated (**, P ≤ 0.01).

    Techniques Used: Flow Cytometry, Mouse Assay, Adoptive Transfer Assay, Binding Assay

    60) Product Images from "Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *"

    Article Title: Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.622274

    Pro-MMP-9 is preferentially recruited via its FN domain to fibroblasts than to the surface of a panel of diverse tumor cell lines. A , cells were incubated in U2OS-conditioned medium containing v5-tagged pro-MMP-9 ( Inputs ), and equal amounts of corresponding membrane fractions ( Membranes ) were loaded onto gels. A representative anti-v5 antibody immunoblot of membrane preparations from the indicated tumor cell lines as well as HSF and MRC-5 fibroblasts from three independent experiments is shown. Transferrin receptor ( Trf ) was used as a membrane equal loading control. B , MMP-9 and its mutants. Shown is a schematic representation of wild type pro-MMP-9; the catalytically dead mutant carrying the E402Q substitution within the catalytic domain; ΔFN, which lacks the FN domain; ΔHEX, which lacks the hemopexin homology domain; FN, which is composed of the fibronectin II domain (FN223–389) only; and HEX, which is composed of the hemopexin homology domain (HEX520–707) only. All constructs were tagged at their 3′ extremity with sequences encoding 6 histidine residues and the v5 peptide. All cDNAs were inserted into the pLIVC retroviral vector and stably expressed in CHO cells. Immunoblots of v5-tagged mutants expressed in U20S transfectant-conditioned culture media are shown ( right panel ). C and D , the FN domain of MMP-9 is necessary and sufficient for its recruitment to the fibroblast cell surface. C , MRC-5 cells were incubated in conditioned culture media from U2OS cells engineered to express recombinant v5-tagged pro-MMP-9 or the different mutants (FN, ΔHEX, ΔFN, HEX, and E402Q ( Inputs )) and lysed, and equal amounts of cell lysates were loaded onto gels. A representative anti-v5 antibody immunoblot of MRC-5 cell membranes from three independent experiments shows that pro-MMP-9 and mutants containing the FN domain (FN, ΔHEX, and E402Q) are recruited to the MRC-5 cell surface, whereas those lacking the FN domain (ΔFN and HEX) are not at all or weakly so. D , MRC-5 cells were incubated in conditioned culture medium of U2OS cells expressing recombinant v5-tagged pro-MMP-9 or the different mutants (FN, E402Q, and ΔFN) and CD5, which was used as negative control, and anti-v5 antibody reactivity with intact cells was assessed by immunofluorescence. Only pro-MMP-9 and mutants containing the FN domain (FN and E402Q) are recruited to the MRC-5 cell surface. DAPI ( blue ) was used to visualize nuclei.
    Figure Legend Snippet: Pro-MMP-9 is preferentially recruited via its FN domain to fibroblasts than to the surface of a panel of diverse tumor cell lines. A , cells were incubated in U2OS-conditioned medium containing v5-tagged pro-MMP-9 ( Inputs ), and equal amounts of corresponding membrane fractions ( Membranes ) were loaded onto gels. A representative anti-v5 antibody immunoblot of membrane preparations from the indicated tumor cell lines as well as HSF and MRC-5 fibroblasts from three independent experiments is shown. Transferrin receptor ( Trf ) was used as a membrane equal loading control. B , MMP-9 and its mutants. Shown is a schematic representation of wild type pro-MMP-9; the catalytically dead mutant carrying the E402Q substitution within the catalytic domain; ΔFN, which lacks the FN domain; ΔHEX, which lacks the hemopexin homology domain; FN, which is composed of the fibronectin II domain (FN223–389) only; and HEX, which is composed of the hemopexin homology domain (HEX520–707) only. All constructs were tagged at their 3′ extremity with sequences encoding 6 histidine residues and the v5 peptide. All cDNAs were inserted into the pLIVC retroviral vector and stably expressed in CHO cells. Immunoblots of v5-tagged mutants expressed in U20S transfectant-conditioned culture media are shown ( right panel ). C and D , the FN domain of MMP-9 is necessary and sufficient for its recruitment to the fibroblast cell surface. C , MRC-5 cells were incubated in conditioned culture media from U2OS cells engineered to express recombinant v5-tagged pro-MMP-9 or the different mutants (FN, ΔHEX, ΔFN, HEX, and E402Q ( Inputs )) and lysed, and equal amounts of cell lysates were loaded onto gels. A representative anti-v5 antibody immunoblot of MRC-5 cell membranes from three independent experiments shows that pro-MMP-9 and mutants containing the FN domain (FN, ΔHEX, and E402Q) are recruited to the MRC-5 cell surface, whereas those lacking the FN domain (ΔFN and HEX) are not at all or weakly so. D , MRC-5 cells were incubated in conditioned culture medium of U2OS cells expressing recombinant v5-tagged pro-MMP-9 or the different mutants (FN, E402Q, and ΔFN) and CD5, which was used as negative control, and anti-v5 antibody reactivity with intact cells was assessed by immunofluorescence. Only pro-MMP-9 and mutants containing the FN domain (FN and E402Q) are recruited to the MRC-5 cell surface. DAPI ( blue ) was used to visualize nuclei.

    Techniques Used: Incubation, Mutagenesis, Construct, Plasmid Preparation, Stable Transfection, Western Blot, Transfection, Recombinant, Expressing, Negative Control, Immunofluorescence

    LH3 provides a cell surface docking mechanism for MMP-9 by recognizing its FN domain. A , mass spectrometry analysis. PLOD3_HUMAN (LH3) appeared to be specifically pulled down by MMP-9 and the FN domain according to mass spectrometry analysis with 95% probability (140% probability variance). Coomassie Blue staining of the pulldown of labeled MMP-9, FN, and ΔFN is shown ( right panel ). B , MMP-9 interacts with endogenous LH3 at the fibroblast cell surface via its FN domain. A representative anti-LH3 antibody immunoblot of anti-v5 antibody immunoprecipitates shows that endogenous LH3 is immunoprecipitated with both v5-tagged MMP-9 and its recombinant FN domain ( v5 IP MMP-9 and FN ), confirming the specificity of the interaction via fibronectin type II-like motifs. Anti-FLAG antibody immunoprecipitations ( IP ) constitute a control. C , interaction between MMP-9 and LH3 decreases upon LH3 depletion. PLA analysis between v5-tagged MMP-9 or ΔFN and endogenous LH3 in HSF showing specificity of the interaction between LH3 and the FN domain of MMP-9 ( left panel ) and impairment of the interaction when LH3 is depleted ( KD ) ( right panel ). nb , number. Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05.
    Figure Legend Snippet: LH3 provides a cell surface docking mechanism for MMP-9 by recognizing its FN domain. A , mass spectrometry analysis. PLOD3_HUMAN (LH3) appeared to be specifically pulled down by MMP-9 and the FN domain according to mass spectrometry analysis with 95% probability (140% probability variance). Coomassie Blue staining of the pulldown of labeled MMP-9, FN, and ΔFN is shown ( right panel ). B , MMP-9 interacts with endogenous LH3 at the fibroblast cell surface via its FN domain. A representative anti-LH3 antibody immunoblot of anti-v5 antibody immunoprecipitates shows that endogenous LH3 is immunoprecipitated with both v5-tagged MMP-9 and its recombinant FN domain ( v5 IP MMP-9 and FN ), confirming the specificity of the interaction via fibronectin type II-like motifs. Anti-FLAG antibody immunoprecipitations ( IP ) constitute a control. C , interaction between MMP-9 and LH3 decreases upon LH3 depletion. PLA analysis between v5-tagged MMP-9 or ΔFN and endogenous LH3 in HSF showing specificity of the interaction between LH3 and the FN domain of MMP-9 ( left panel ) and impairment of the interaction when LH3 is depleted ( KD ) ( right panel ). nb , number. Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05.

    Techniques Used: Mass Spectrometry, Staining, Labeling, Immunoprecipitation, Recombinant, Proximity Ligation Assay

    FACS analysis of v5-tagged MMP-9 variant recruitment to living MRC-5 cells. A , live MRC-5 cells incubated with v5-tagged MMP-9 were stained with mouse anti-v5 or an irrelevant mouse isotype-matched antibody followed by anti-mouse Alexa Fluor 488-conjugated antibody. Single cells (69.9) were gated after doublet exclusion, and DAPI-negative cells (42.5) were considered to be live. The shift in the histogram between isotype-matched (0.7) and anti-v5 antibody (29.0) profiles shows MMP-9 presence on the MRC-5 cell surface. B , live MRC-5 cells incubated with v5-tagged ΔFN were stained with anti-v5 or an irrelevant mouse isotype-matched antibody followed by Alexa Fluor 488-conjugated anti-mouse antibody. No shift between isotype-matched (0.83) and anti-v5 antibody peaks (5.89) is observed. C , superposition of isotype-matched ( orange ) and anti-v5 antibody (ΔFN, blue ; MMP-9, green ) staining profiles. SS-A , side scatter area; FS-A , forward scatter area; FS-W , forward scatter width.
    Figure Legend Snippet: FACS analysis of v5-tagged MMP-9 variant recruitment to living MRC-5 cells. A , live MRC-5 cells incubated with v5-tagged MMP-9 were stained with mouse anti-v5 or an irrelevant mouse isotype-matched antibody followed by anti-mouse Alexa Fluor 488-conjugated antibody. Single cells (69.9) were gated after doublet exclusion, and DAPI-negative cells (42.5) were considered to be live. The shift in the histogram between isotype-matched (0.7) and anti-v5 antibody (29.0) profiles shows MMP-9 presence on the MRC-5 cell surface. B , live MRC-5 cells incubated with v5-tagged ΔFN were stained with anti-v5 or an irrelevant mouse isotype-matched antibody followed by Alexa Fluor 488-conjugated anti-mouse antibody. No shift between isotype-matched (0.83) and anti-v5 antibody peaks (5.89) is observed. C , superposition of isotype-matched ( orange ) and anti-v5 antibody (ΔFN, blue ; MMP-9, green ) staining profiles. SS-A , side scatter area; FS-A , forward scatter area; FS-W , forward scatter width.

    Techniques Used: FACS, Variant Assay, Incubation, Staining

    Depletion of LH3 decreases MMP-9 cell surface recruitment as does the recombinant FN domain, which displaces MMP-9 from the fibroblast membrane. A , LH3 down-regulation in MRC-5 decreases MMP-9 cell surface recruitment. Equal amounts of cell lysates ( CL ) from control ( ctl ) and LH3-depleted ( KD ) MRC-5 incubated with v5-tagged MMP-9 ( SN ) were loaded onto gels. Recruitment was quantified by densitometry. A representative anti-v5 antibody immunoblot of equal amounts of MRC-5 cell lysates from three independent experiments ( lower panel ) and the corresponding anti-LH3 antibody immunoblot are shown. B , PLA analysis of WT or LH3-depleted HSF treated with v5-tagged MMP-9. The histogram shows interaction between v5-tagged MMP-9 and endogenous LH3 that is decreased when LH3 is depleted. Lower panels show immunofluorescence images of MMP-9 recruitment ( green ) to membranes of HSF with an overlap between MMP-9 and LH3 ( yellow ) that is decreased by LH3 depletion. C , the FN domain prevents MMP-9/LH3 interaction in a dose-dependent manner. PLA analysis of MRC-5 cells treated with MMP-9 only, FN only, or MMP-9 with increased concentrations of the FN domain (1:1, 1:2, and 1:10 corresponding to 1:3.4, 1:6.8, and 1:34 molar ratios). The histogram shows interaction between MMP-9 and endogenous LH3 that decreases as the concentration of FN increases. nb , number. Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
    Figure Legend Snippet: Depletion of LH3 decreases MMP-9 cell surface recruitment as does the recombinant FN domain, which displaces MMP-9 from the fibroblast membrane. A , LH3 down-regulation in MRC-5 decreases MMP-9 cell surface recruitment. Equal amounts of cell lysates ( CL ) from control ( ctl ) and LH3-depleted ( KD ) MRC-5 incubated with v5-tagged MMP-9 ( SN ) were loaded onto gels. Recruitment was quantified by densitometry. A representative anti-v5 antibody immunoblot of equal amounts of MRC-5 cell lysates from three independent experiments ( lower panel ) and the corresponding anti-LH3 antibody immunoblot are shown. B , PLA analysis of WT or LH3-depleted HSF treated with v5-tagged MMP-9. The histogram shows interaction between v5-tagged MMP-9 and endogenous LH3 that is decreased when LH3 is depleted. Lower panels show immunofluorescence images of MMP-9 recruitment ( green ) to membranes of HSF with an overlap between MMP-9 and LH3 ( yellow ) that is decreased by LH3 depletion. C , the FN domain prevents MMP-9/LH3 interaction in a dose-dependent manner. PLA analysis of MRC-5 cells treated with MMP-9 only, FN only, or MMP-9 with increased concentrations of the FN domain (1:1, 1:2, and 1:10 corresponding to 1:3.4, 1:6.8, and 1:34 molar ratios). The histogram shows interaction between MMP-9 and endogenous LH3 that decreases as the concentration of FN increases. nb , number. Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

    Techniques Used: Recombinant, CTL Assay, Incubation, Proximity Ligation Assay, Immunofluorescence, Concentration Assay

    MMP-9 activity promotes latent TGF-β activation and induces α-SMA expression in cultured fibroblasts. A , active MMP-9 is recruited to fibroblast cell surface. MRC-5 cells were incubated in conditioned culture media ( SN ) containing 0.5 μg/ml of purified pro- or active MMP-9 or the ΔFN mutant, and equal amounts of corresponding cell lysates ( CL ) were loaded onto gels. Recruitment to the MRC-5 cell surface was verified by anti-v5 antibody immunoblotting. B , MMP-9 activity promotes TGF-β activation in cultured MRC-5 cells. Conditioned culture medium of MRC-5 cells incubated for 72 h with 0.5 μg/ml pro-MMP-9, active MMP-9, E402Q, ΔFN, or TGF-β1 (10 ng/ml) was collected for luciferase assays using TGF-β-responsive TMLC. Luminescence reflecting TGF-β activity was quantified by relative light units ( RLU ). C , MMP-9 induces α-SMA expression in resting MRC-5 cells. MRC-5 cells were incubated for 72 h with 0.5 μg/ml pro-MMP-9, active MMP-9, E402Q, ΔFN, or TGF-β1 (10 ng/ml). A representative anti-α-SMA antibody immunoblot of equal amounts of MRC-5 cell lysates from four independent experiments ( upper panel ) is shown. Analysis of α-SMA expression from four independent experiments ( lower panel ) is shown. Expression quantification was normalized to tubulin ( tub ). Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05; ****, p ≤ 0.0001.
    Figure Legend Snippet: MMP-9 activity promotes latent TGF-β activation and induces α-SMA expression in cultured fibroblasts. A , active MMP-9 is recruited to fibroblast cell surface. MRC-5 cells were incubated in conditioned culture media ( SN ) containing 0.5 μg/ml of purified pro- or active MMP-9 or the ΔFN mutant, and equal amounts of corresponding cell lysates ( CL ) were loaded onto gels. Recruitment to the MRC-5 cell surface was verified by anti-v5 antibody immunoblotting. B , MMP-9 activity promotes TGF-β activation in cultured MRC-5 cells. Conditioned culture medium of MRC-5 cells incubated for 72 h with 0.5 μg/ml pro-MMP-9, active MMP-9, E402Q, ΔFN, or TGF-β1 (10 ng/ml) was collected for luciferase assays using TGF-β-responsive TMLC. Luminescence reflecting TGF-β activity was quantified by relative light units ( RLU ). C , MMP-9 induces α-SMA expression in resting MRC-5 cells. MRC-5 cells were incubated for 72 h with 0.5 μg/ml pro-MMP-9, active MMP-9, E402Q, ΔFN, or TGF-β1 (10 ng/ml). A representative anti-α-SMA antibody immunoblot of equal amounts of MRC-5 cell lysates from four independent experiments ( upper panel ) is shown. Analysis of α-SMA expression from four independent experiments ( lower panel ) is shown. Expression quantification was normalized to tubulin ( tub ). Results represent mean values ±S.E. ( error bars ). *, p ≤ 0.05; ****, p ≤ 0.0001.

    Techniques Used: Activity Assay, Activation Assay, Expressing, Cell Culture, Incubation, Purification, Mutagenesis, Luciferase

    61) Product Images from "Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody"

    Article Title: Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054509

    Comparison of Y-142 and Y-073 biological activities toward sHB-EGF. A. Binding activity of Y-142 and Y-073 toward sHB-EGF. Anti-HB-EGF antibody was incubated at various concentrations in an sHB-EGF-coated or a BSA-coated plate. Antibody binding was detected with an HRP-labeled anti-mouse IgG antibody. Y-142 binding to BSA, white square; Y-073 binding to BSA, white circle; Y-142 binding to sHB-EGF, black square; Y-073 binding to sHB-EGF, black circle. The data points represent the mean ± standard deviation (SD) of values acquired in duplicate. B. Neutralizing activity of Y-142 and Y-073 against sHB-EGF-induced EGFR phosphorylation. Anti-HB-EGF antibody was incubated at the indicated concentrations in the presence of 10 nM sHB-EGF with NUGC-3 cells for 15 minutes at 37°C. Cell lysates were incubated on an anti-EGFR antibody-coated plate, followed by incubation with HRP-labeled anti-phosphotyrosine antibody. No sHB-EGF, white circle; sHB-EGF and control IgG, white square; sHB-EGF and Y-142, black square; sHB-EGF and Y-073, black circle. The data points represent the mean ± SD of values acquired in triplicate.
    Figure Legend Snippet: Comparison of Y-142 and Y-073 biological activities toward sHB-EGF. A. Binding activity of Y-142 and Y-073 toward sHB-EGF. Anti-HB-EGF antibody was incubated at various concentrations in an sHB-EGF-coated or a BSA-coated plate. Antibody binding was detected with an HRP-labeled anti-mouse IgG antibody. Y-142 binding to BSA, white square; Y-073 binding to BSA, white circle; Y-142 binding to sHB-EGF, black square; Y-073 binding to sHB-EGF, black circle. The data points represent the mean ± standard deviation (SD) of values acquired in duplicate. B. Neutralizing activity of Y-142 and Y-073 against sHB-EGF-induced EGFR phosphorylation. Anti-HB-EGF antibody was incubated at the indicated concentrations in the presence of 10 nM sHB-EGF with NUGC-3 cells for 15 minutes at 37°C. Cell lysates were incubated on an anti-EGFR antibody-coated plate, followed by incubation with HRP-labeled anti-phosphotyrosine antibody. No sHB-EGF, white circle; sHB-EGF and control IgG, white square; sHB-EGF and Y-142, black square; sHB-EGF and Y-073, black circle. The data points represent the mean ± SD of values acquired in triplicate.

    Techniques Used: Binding Assay, Activity Assay, Incubation, Labeling, Standard Deviation

    62) Product Images from "Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2"

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    Journal: Veterinary Research

    doi: 10.1051/vetres/2010048

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )
    Figure Legend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Techniques Used: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing

    63) Product Images from "CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1"

    Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1

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

    doi:

    Eliminating IFNγ and IL-4 does not induce donor-reactive Th17. IFNγ-/- allograft recipients were either left untreated ( A ) or treated with neutralizing anti-IL-4 mAb ( B ). At the time of rejection (day 7 post-transplant), splenocytes were
    Figure Legend Snippet: Eliminating IFNγ and IL-4 does not induce donor-reactive Th17. IFNγ-/- allograft recipients were either left untreated ( A ) or treated with neutralizing anti-IL-4 mAb ( B ). At the time of rejection (day 7 post-transplant), splenocytes were

    Techniques Used:

    CD8+ T cells produce IFNγ and IL-17 in T-bet-/- allograft recipients. Splenocytes were harvested from T-bet-/- allograft recipients on day 7 post-transplant. Splenocytes were either left untreated (closed squares) or were depleted of CD4+ (open
    Figure Legend Snippet: CD8+ T cells produce IFNγ and IL-17 in T-bet-/- allograft recipients. Splenocytes were harvested from T-bet-/- allograft recipients on day 7 post-transplant. Splenocytes were either left untreated (closed squares) or were depleted of CD4+ (open

    Techniques Used:

    64) Product Images from "Characterization of Glutamatergic Neurons in the Rat Atrial Intrinsic Cardiac Ganglia that Project to the Cardiac Ventricular Wall"

    Article Title: Characterization of Glutamatergic Neurons in the Rat Atrial Intrinsic Cardiac Ganglia that Project to the Cardiac Ventricular Wall

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2016.05.002

    (A–C) : GLS-immunoreactive (IR) neurons in the retrograde labeling study. A : GLS-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open circles indicate non-retrogradely labeled neurons and filled circles indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong GLS-immunoreactivity (ir). Neurons with moderate and strong GLS-ir were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 400.7µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 282.6µm 2 . C : One way ANOVA compared the mean cell areas of retrogradely labeled neurons (Mean ± SEM: 387.2± 21.7µm 2 ), non-retrogradely labeled neurons (Mean ± SEM: 276.4 ±17.2µm 2 ) and total neurons (Mean ± SEM: 284.8±16.5µm 2 ). Retrogradely labeled neurons were statistically larger than the total population and the non-retrogradely labeled neurons (P value = 0.004).
    Figure Legend Snippet: (A–C) : GLS-immunoreactive (IR) neurons in the retrograde labeling study. A : GLS-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open circles indicate non-retrogradely labeled neurons and filled circles indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong GLS-immunoreactivity (ir). Neurons with moderate and strong GLS-ir were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 400.7µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 282.6µm 2 . C : One way ANOVA compared the mean cell areas of retrogradely labeled neurons (Mean ± SEM: 387.2± 21.7µm 2 ), non-retrogradely labeled neurons (Mean ± SEM: 276.4 ±17.2µm 2 ) and total neurons (Mean ± SEM: 284.8±16.5µm 2 ). Retrogradely labeled neurons were statistically larger than the total population and the non-retrogradely labeled neurons (P value = 0.004).

    Techniques Used: Labeling, Whole Genome Amplification, Significance Assay

    (A–D) : VGLUT2-IR neurons in the retrograde labeling study. A : VGLUT2-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open hexagons indicate non-retrogradely labeled neurons and filled hexagons indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong VGLUT2-ir. Neurons with moderate and strong immunoreactivity were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 399.25 µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 304.03µm 2 . C : One way ANOVA compared the mean cell area of retrogradely labeled neurons (Mean± SEM: 396.4 ± 45.7µm 2 ), non-retrogradely labeled neurons (Mean± SEM: 298.9 ± 16.8µm 2 ) and total neurons (Mean± SEM: 307.2 ±18.4 µm 2 ). Retrogradely labeled neurons show a trend of increase in size, but the mean cell sizes were distributed widely in the retrogradely labeled group, so statistical significance was not achieved. (P value= 0.113). D: The average sizes of retrogradely labeled, GLS-, VGLUT2, and VGLUT1-IR neurons were compared with one way ANOVA. Although the VGLUT1-IR group appeared larger, there was no significance difference in the groups (P value = 0.14).
    Figure Legend Snippet: (A–D) : VGLUT2-IR neurons in the retrograde labeling study. A : VGLUT2-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open hexagons indicate non-retrogradely labeled neurons and filled hexagons indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong VGLUT2-ir. Neurons with moderate and strong immunoreactivity were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 399.25 µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 304.03µm 2 . C : One way ANOVA compared the mean cell area of retrogradely labeled neurons (Mean± SEM: 396.4 ± 45.7µm 2 ), non-retrogradely labeled neurons (Mean± SEM: 298.9 ± 16.8µm 2 ) and total neurons (Mean± SEM: 307.2 ±18.4 µm 2 ). Retrogradely labeled neurons show a trend of increase in size, but the mean cell sizes were distributed widely in the retrogradely labeled group, so statistical significance was not achieved. (P value= 0.113). D: The average sizes of retrogradely labeled, GLS-, VGLUT2, and VGLUT1-IR neurons were compared with one way ANOVA. Although the VGLUT1-IR group appeared larger, there was no significance difference in the groups (P value = 0.14).

    Techniques Used: Labeling, Whole Genome Amplification, Significance Assay

    (A–I) : Retrograde labeling and immunohistochemistry for rat ICG neurons (arrows). A–C : GLS-IR neurons in rat ICG. D–F : VGLUT1-IR neurons in rat ICG (red). G–I : VGLUT2-IR neurons in rat ICG (red). B, E, H : WGA-HRP retrogradely labeled cells (Green); C, F, I : Merged pictures of GLS, VGLUT1, or VGLUT2 staining and WGA-HRP staining. DAPI staining (blue) also was performed to show individual cell nuclei.
    Figure Legend Snippet: (A–I) : Retrograde labeling and immunohistochemistry for rat ICG neurons (arrows). A–C : GLS-IR neurons in rat ICG. D–F : VGLUT1-IR neurons in rat ICG (red). G–I : VGLUT2-IR neurons in rat ICG (red). B, E, H : WGA-HRP retrogradely labeled cells (Green); C, F, I : Merged pictures of GLS, VGLUT1, or VGLUT2 staining and WGA-HRP staining. DAPI staining (blue) also was performed to show individual cell nuclei.

    Techniques Used: Labeling, Immunohistochemistry, Whole Genome Amplification, Staining

    (A–C) : VGLUT1-IR neurons in the retrograde labeling study. A : VGLUT1-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open diamonds indicate non-retrogradely labeled neurons and filled diamonds indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong VGLUT1-immunoreactivity (ir). Neurons with moderate and strong VGLUT1-ir were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 493.0 µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 314.7 µm 2 . C : One way ANOVA test compared the mean cell areas of retrogradely labeled neurons (Mean ± SEM: 476.9± 21.6µm 2 ), non-retrogradely labeled neurons (Mean ± SEM: 316.0 ± 25.6µm 2 ) and total neurons (Mean ± SEM: 326.4 ± 29.7 µm 2 ). Retrogradely labeled neurons were statistically larger than the total population and the non-retrogradely labeled neurons (P value= 0.008).
    Figure Legend Snippet: (A–C) : VGLUT1-IR neurons in the retrograde labeling study. A : VGLUT1-IR neurons from the WGA-HRP retrograde labeling study were plotted by area vs. MGI. Open diamonds indicate non-retrogradely labeled neurons and filled diamonds indicate retrogradely labeled neurons. Neurons were classified into groups of low, moderate, and strong VGLUT1-immunoreactivity (ir). Neurons with moderate and strong VGLUT1-ir were chosen for subsequent analysis. B : Histogram distribution of WGA-HRP retrogradely labeled neurons and non-retrogradely labeled neurons. The mean neuronal profile of retrogradely labeled neurons was 493.0 µm 2 and the mean neuronal profile of non-retrogradely labeled neurons was 314.7 µm 2 . C : One way ANOVA test compared the mean cell areas of retrogradely labeled neurons (Mean ± SEM: 476.9± 21.6µm 2 ), non-retrogradely labeled neurons (Mean ± SEM: 316.0 ± 25.6µm 2 ) and total neurons (Mean ± SEM: 326.4 ± 29.7 µm 2 ). Retrogradely labeled neurons were statistically larger than the total population and the non-retrogradely labeled neurons (P value= 0.008).

    Techniques Used: Labeling, Whole Genome Amplification, Significance Assay

    65) Product Images from "?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling"

    Article Title: ?-Catenin Phosphorylated at Serine 45 Is Spatially Uncoupled from ?-Catenin Phosphorylated in the GSK3 Domain: Implications for Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010184

    Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.
    Figure Legend Snippet: Cytosolic N-terminally unphosphorylated β-catenin is primarily monomeric. A detergent-free cytosolic fraction from SW480 control (A) or α-catenin shRNA knock-down (B) cells was subjected to gel filtration chromatography and immunoblot analysis. A) ABC sizes as a monomer (compared to calibration standards (not shown) and purified β-catenin ( Fig. S1 ); peak fraction #42), while total β-catenin sizes evenly between monomer- and β-catenin/α-catenin dimer fractions (peak fraction #36). Peak fractions are marked with arrows. B–D) β-catenin dimer fraction is due to association with α-catenin. B) Size fractionation of cytosol from SW480 cells depleted of α-catenin by shRNA. C) Immunoblot of SW480 control and α-catenin knock down lysates. D) [ 35 S]-methionine/cysteine-labeling of SW480 cells and immunoprecipitation of α-catenin and β-catenin (1∶100, lanes 3 and 5; 1∶300, lanes 4 and 6). Autoradiogram reveals the major binding partner of β-catenin in this cell type is α-catenin. No antibody (lane 1) or non-immune control (lane 2) are also shown.

    Techniques Used: shRNA, Filtration, Chromatography, Purification, Fractionation, Labeling, Immunoprecipitation, Binding Assay

    66) Product Images from "Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes"

    Article Title: Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes

    Journal: Nature Communications

    doi: 10.1038/ncomms14286

    Profiling cellular substrates of Uba6- and Uba1-dependent ubiquitination. ( a ) Flow chart of procedures to identify xUba6- and xUba1-dependent ubiquitination substrates by tandem affinity purification and proteomic procedures. ( b ) Specific reactions of the xUB-xUba1 pair and the xUB-xUba6 pair in HEK293 cells with lentiviral transduction. Upper panels indicate that HBT(histidine/biotinylation-signal tag)-xUB physically conjugates with FLAG-xUba6 and FLAG-xUba1, whereas HBT-wt UB shows no conjugation with FLAG-xUba6 or FLAG-xUba1. HEK293 cells were infected with recombinant lentiviruses for expression of the indicated proteins, followed by drug selection for stable integration. Conjugation of wt UB or xUB with wt E1 or xE1 proteins was examined by immunoprecipitation for E1 proteins under a non-reducing condition, followed by immunoblotting for the poly-histidine tag of UB (the upper panel) or for the biotinylation tag with streptavidin conjugated with horse radish peroxidase (HRP) (the middle panel). The arrow indicates UB proteins, while the asterisk shows a common protein around 25 kDa with cross-reactivity for the anti-penta-histidine antibody. The bottom panels demonstrate total expression levels of each protein determined by direct immunoblotting for the indicated epitope tag or tubulin as a loading control. Ctrl, parental HEK293 cells without viral transduction; GFP, cells infected with a lentivirus for green fluorescent protein. ( c ) Numbers of Uba6-specific, Uba1-specific and Uba6/Uba1-shared ubiquitination substrates identified by the OUT screen. ( d ) E2 enzymes conjugated with HBT-xUB in xUba6- or xUba1-dependent manners. The enzymes shown in the centre were identified by both xUba6- and xUba1-mediated screens.
    Figure Legend Snippet: Profiling cellular substrates of Uba6- and Uba1-dependent ubiquitination. ( a ) Flow chart of procedures to identify xUba6- and xUba1-dependent ubiquitination substrates by tandem affinity purification and proteomic procedures. ( b ) Specific reactions of the xUB-xUba1 pair and the xUB-xUba6 pair in HEK293 cells with lentiviral transduction. Upper panels indicate that HBT(histidine/biotinylation-signal tag)-xUB physically conjugates with FLAG-xUba6 and FLAG-xUba1, whereas HBT-wt UB shows no conjugation with FLAG-xUba6 or FLAG-xUba1. HEK293 cells were infected with recombinant lentiviruses for expression of the indicated proteins, followed by drug selection for stable integration. Conjugation of wt UB or xUB with wt E1 or xE1 proteins was examined by immunoprecipitation for E1 proteins under a non-reducing condition, followed by immunoblotting for the poly-histidine tag of UB (the upper panel) or for the biotinylation tag with streptavidin conjugated with horse radish peroxidase (HRP) (the middle panel). The arrow indicates UB proteins, while the asterisk shows a common protein around 25 kDa with cross-reactivity for the anti-penta-histidine antibody. The bottom panels demonstrate total expression levels of each protein determined by direct immunoblotting for the indicated epitope tag or tubulin as a loading control. Ctrl, parental HEK293 cells without viral transduction; GFP, cells infected with a lentivirus for green fluorescent protein. ( c ) Numbers of Uba6-specific, Uba1-specific and Uba6/Uba1-shared ubiquitination substrates identified by the OUT screen. ( d ) E2 enzymes conjugated with HBT-xUB in xUba6- or xUba1-dependent manners. The enzymes shown in the centre were identified by both xUba6- and xUba1-mediated screens.

    Techniques Used: Flow Cytometry, Affinity Purification, Transduction, Conjugation Assay, Infection, Recombinant, Expressing, Selection, Immunoprecipitation

    67) Product Images from "ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons"

    Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1409-12.2012

    ARF6 regulates β1 integrin localization in DRG axons and growth cones. Images are axons and growth cones of adult DRG neurons. ARF6 and β1 integrins partly colocalize in distinct regions (triangle) and vesicular structures (arrows) ( A , maximum projection and single confocal section inset). Overlap is also present after expression of wt ARF6-HA in growth cone vesicles ( B , arrows; inset is a single confocal section) and filopodia ( B , dotted lines). Expression of dominant-negative ARF6 T27N leads to prominent β1 integrin localization at the growth cone periphery, where the two proteins colocalize ( C , dotted lines). Colocalization is also evident in vesicles ( C , arrows; inset is a single confocal section). Expression of constitutively active ARF6 Q67L results in accumulation of ARF6 in swollen intracellular structures within the growth cone, which also label for β1 integrin ( D , circled), and colocalization is also apparent at vesicular structures of more usual size ( D , arrows). Swollen vesicles containing ARF6 and β1 integrin were also present in DRG axons ( E , inset is a single confocal section). Subcellular fractionation confirmed that ARF6 is enriched in the endosomal fraction (fraction 5) in maturely differentiated PC12 cells, as is β1 integrin and Rab11 ( F ). Scale bars: 10 μm. A–C and E are maximum projections with a single confocal slice magnified in the inset. D is a single confocal section.
    Figure Legend Snippet: ARF6 regulates β1 integrin localization in DRG axons and growth cones. Images are axons and growth cones of adult DRG neurons. ARF6 and β1 integrins partly colocalize in distinct regions (triangle) and vesicular structures (arrows) ( A , maximum projection and single confocal section inset). Overlap is also present after expression of wt ARF6-HA in growth cone vesicles ( B , arrows; inset is a single confocal section) and filopodia ( B , dotted lines). Expression of dominant-negative ARF6 T27N leads to prominent β1 integrin localization at the growth cone periphery, where the two proteins colocalize ( C , dotted lines). Colocalization is also evident in vesicles ( C , arrows; inset is a single confocal section). Expression of constitutively active ARF6 Q67L results in accumulation of ARF6 in swollen intracellular structures within the growth cone, which also label for β1 integrin ( D , circled), and colocalization is also apparent at vesicular structures of more usual size ( D , arrows). Swollen vesicles containing ARF6 and β1 integrin were also present in DRG axons ( E , inset is a single confocal section). Subcellular fractionation confirmed that ARF6 is enriched in the endosomal fraction (fraction 5) in maturely differentiated PC12 cells, as is β1 integrin and Rab11 ( F ). Scale bars: 10 μm. A–C and E are maximum projections with a single confocal slice magnified in the inset. D is a single confocal section.

    Techniques Used: Expressing, Dominant Negative Mutation, Fractionation

    68) Product Images from "Antibody induction of lupus-like neuropsychiatric manifestations"

    Article Title: Antibody induction of lupus-like neuropsychiatric manifestations

    Journal: Journal of neuroimmunology

    doi: 10.1016/j.jneuroim.2006.10.017

    SB31 recognizes the brain protein dynamin-1. A BALB/cByJ mouse brain homogenate was immunoprecipitated with NB28, a monoclonal anti-dynamin-1 (OncogeneResearch Products, Boston, MA), and the blotted proteins were probed with SB31 followed by biotinylated goat anti-mouse IgG and streptavidin-HRP. The arrow indicates dynamin-1.
    Figure Legend Snippet: SB31 recognizes the brain protein dynamin-1. A BALB/cByJ mouse brain homogenate was immunoprecipitated with NB28, a monoclonal anti-dynamin-1 (OncogeneResearch Products, Boston, MA), and the blotted proteins were probed with SB31 followed by biotinylated goat anti-mouse IgG and streptavidin-HRP. The arrow indicates dynamin-1.

    Techniques Used: Immunoprecipitation

    69) Product Images from "Lung-derived innate cytokines: new epigenetic targets of allergen-specific sublingual immunotherapy"

    Article Title: Lung-derived innate cytokines: new epigenetic targets of allergen-specific sublingual immunotherapy

    Journal: Iranian Journal of Basic Medical Sciences

    doi:

    Systemic responses after sublingual immunotherapy. a-c, Specific IgE (a), IgG1 (b), and IgG2a (c) serum levels in control (non-sensitized), PBS-treated (sham treated), and rChe a 2-treated groups. d-h, IFN-γ (d), IL-4 (e), TGF-β (g), and IL-10 (h) levels and IFN-γ/IL-4 (f) in the supernatants of splenocytes stimulated with rChe a 2 from the respective groups. Bars indicate the average OD of antibody levels or cytokine concentrations in pg/ml ± SEM. ns, not significant. * P
    Figure Legend Snippet: Systemic responses after sublingual immunotherapy. a-c, Specific IgE (a), IgG1 (b), and IgG2a (c) serum levels in control (non-sensitized), PBS-treated (sham treated), and rChe a 2-treated groups. d-h, IFN-γ (d), IL-4 (e), TGF-β (g), and IL-10 (h) levels and IFN-γ/IL-4 (f) in the supernatants of splenocytes stimulated with rChe a 2 from the respective groups. Bars indicate the average OD of antibody levels or cytokine concentrations in pg/ml ± SEM. ns, not significant. * P

    Techniques Used:

    70) Product Images from "Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder"

    Article Title: Sulforaphane Improves Ischemia-Induced Detrusor Overactivity by Downregulating the Enhancement of Associated Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Rat Bladder

    Journal: Scientific Reports

    doi: 10.1038/srep36110

    Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p
    Figure Legend Snippet: Changes in bladder expression of ( A ) c-Nrf2, ( B ) Keap1, ( C ) n-Nrf2, ( D ) GRP78 (ER stress), ( E ) CHOP (ER stress), ( F ) caspase 3, ( G ) Beclin-1, ( H ) p62, ( I ) LC3 II, ( J ) muscarinic M 2 receptor, ( K ) muscarinic M 3 receptor, ( L ) purinergic P 2 X 1 receptor, ( M ) purinergic P 2 X 2 receptor, and ( N ) purinergic P 2 X 3 receptor in response to 2WBI, 4WBI, and treatments. All of the experiments were performed in three rats of each group. * p

    Techniques Used: Expressing

    Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p
    Figure Legend Snippet: Representative histological findings in response to bladder ischemia. The figure shows ( A – C ) GRP78 staining, ( D – F ) CHOP staining, ( G – I ) Beclin-1 staining, ( J – L ) LC3 II, and ( M – O ) TUNEL staining in the sham, 2WBI, and 4WBI groups. All images are magnified with x400. The scale bar is 50 μm. Respective statistic data are shown in ( P ) GRP78, ( Q ) CHOP, ( R ) Beclin-1, ( S ) LC3 II and ( T ) TUNEL stain (n = 6 in each test) in each group. * p

    Techniques Used: Staining, TUNEL Assay

    71) Product Images from "IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production"

    Article Title: IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production

    Journal: Blood

    doi: 10.1182/blood-2009-07-230631

    Peritoneal macrophages from SHIP −/− mice produce IL-6 . (A) IL-6 production of freshly isolated and purified macrophages from bone marrow, spleen, lung, and peritoneum of WT and SHIP −/− mice, analyzed by ELISA after a 6-hour culture. Data are mean ± SE of 3 identical experiments. (B) Subpopulations of peritoneal cells were identified by flow cytometry as macrophages (Mac-1 + , F4/80 + ), monocytes (Mac-1 + , F4/80 − , Ly-6C hi ), myeloid dendritic cells (CD11c + , Ly-6C + ), and neutrophils (Mac-1 + , Gr-1 + , Ly-6C + ) and analyzed for intracellular IL-6. Shown is the total number of each population that expressed intracellular IL-6. Data are mean ± SE of 3 animals. (C) IL-6 histogram of peritoneal macrophages (defined as Mac-1 + , F4/80 + ) from WT (dashed line) or SHIP −/− (solid line) mice. Unstained cells are shown in gray. (D) Peritoneal macrophages identified with anti–Mac-1 and anti-F4/80 antibodies. (E) Total cells from 7 separate animals.
    Figure Legend Snippet: Peritoneal macrophages from SHIP −/− mice produce IL-6 . (A) IL-6 production of freshly isolated and purified macrophages from bone marrow, spleen, lung, and peritoneum of WT and SHIP −/− mice, analyzed by ELISA after a 6-hour culture. Data are mean ± SE of 3 identical experiments. (B) Subpopulations of peritoneal cells were identified by flow cytometry as macrophages (Mac-1 + , F4/80 + ), monocytes (Mac-1 + , F4/80 − , Ly-6C hi ), myeloid dendritic cells (CD11c + , Ly-6C + ), and neutrophils (Mac-1 + , Gr-1 + , Ly-6C + ) and analyzed for intracellular IL-6. Shown is the total number of each population that expressed intracellular IL-6. Data are mean ± SE of 3 animals. (C) IL-6 histogram of peritoneal macrophages (defined as Mac-1 + , F4/80 + ) from WT (dashed line) or SHIP −/− (solid line) mice. Unstained cells are shown in gray. (D) Peritoneal macrophages identified with anti–Mac-1 and anti-F4/80 antibodies. (E) Total cells from 7 separate animals.

    Techniques Used: Mouse Assay, Isolation, Purification, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    SHIP −/− mice had a high level of total and aggregated IgG in peritoneum . (A) Total IgG obtained by ELISA in peritoneal lavages of WT and SHIP −/− mice. (B) The lavage fluid was centrifuged 55 000 g , and IgG (defined as aggregated IgG) was measured in the pellet. (A-B) The data were obtained from 6 individual mice, and each point represents a single animal. (C) IgG production from peritoneal cells was determined by ELISA. The F4/80 + macrophages were removed, and the remaining cells were cultured for 24 hours. Cells were collected, and secreted IgG was measured by ELISA from 9 individual mice.
    Figure Legend Snippet: SHIP −/− mice had a high level of total and aggregated IgG in peritoneum . (A) Total IgG obtained by ELISA in peritoneal lavages of WT and SHIP −/− mice. (B) The lavage fluid was centrifuged 55 000 g , and IgG (defined as aggregated IgG) was measured in the pellet. (A-B) The data were obtained from 6 individual mice, and each point represents a single animal. (C) IgG production from peritoneal cells was determined by ELISA. The F4/80 + macrophages were removed, and the remaining cells were cultured for 24 hours. Cells were collected, and secreted IgG was measured by ELISA from 9 individual mice.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

    72) Product Images from "Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells"

    Article Title: Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells

    Journal: Journal of medical entomology

    doi:

    VCAM-1 expression by endothelial cells in response to varying doses of C. arcuatus and T. putrescentiae extracts. Cells were challenged for 6, 12, or 24 h with extract alone or along with TNF α . (Online figure in color.)
    Figure Legend Snippet: VCAM-1 expression by endothelial cells in response to varying doses of C. arcuatus and T. putrescentiae extracts. Cells were challenged for 6, 12, or 24 h with extract alone or along with TNF α . (Online figure in color.)

    Techniques Used: Expressing

    73) Product Images from "Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors"

    Article Title: Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18429-0

    Fibronectin and fibulin-1 deposition by immortalised asthmatic and non-asthmatic ASM cells. FN and FBLN-1 deposition from immortalised non-asthmatic (n = 5) and asthmatic (n = 6) ASM cells after 48 hrs ( a and b respectively) or 72 hrs ( c and d respectively) in response to treatment with 10 ng/ml TGF-β. All data are presented as the mean +/− SEM. Statistical analysis via a two way ANOVA with Sidak’s multiple comparisons test was performed to test differences between control and treatment groups and immortalised asthmatic and non-asthmatic ASM cells. Significance as a result of treatment is represented by **p
    Figure Legend Snippet: Fibronectin and fibulin-1 deposition by immortalised asthmatic and non-asthmatic ASM cells. FN and FBLN-1 deposition from immortalised non-asthmatic (n = 5) and asthmatic (n = 6) ASM cells after 48 hrs ( a and b respectively) or 72 hrs ( c and d respectively) in response to treatment with 10 ng/ml TGF-β. All data are presented as the mean +/− SEM. Statistical analysis via a two way ANOVA with Sidak’s multiple comparisons test was performed to test differences between control and treatment groups and immortalised asthmatic and non-asthmatic ASM cells. Significance as a result of treatment is represented by **p

    Techniques Used:

    74) Product Images from "Human IgE against the major allergen Bet v 1 – defining an epitope with limited cross-reactivity between different PR-10 family proteins"

    Article Title: Human IgE against the major allergen Bet v 1 – defining an epitope with limited cross-reactivity between different PR-10 family proteins

    Journal: Clinical and Experimental Allergy

    doi: 10.1111/cea.12230

    Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.
    Figure Legend Snippet: Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.

    Techniques Used: Sequencing, Clone Assay, Inhibition, Binding Assay, Modification, Mutagenesis, Standard Deviation

    75) Product Images from "Estradiol induces JNK-dependent apoptosis in glioblastoma cells"

    Article Title: Estradiol induces JNK-dependent apoptosis in glioblastoma cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2011.385

    Estradiol inhibited cell proliferation in C6 and T98G cells. Cells were pretreated with SP600125 (20 μM) or vehicle (control) for 30 min, and incubated for 18 h in the presence or absence of estradiol (20 μM) under low growth-stimulated conditions. BrdU incorporation in C6 cells (upper panel) and PCNA expression analysis in T98G cells (lower panel) were carried out. Images were captured using a brightfield microscope at a magnification of ×600.
    Figure Legend Snippet: Estradiol inhibited cell proliferation in C6 and T98G cells. Cells were pretreated with SP600125 (20 μM) or vehicle (control) for 30 min, and incubated for 18 h in the presence or absence of estradiol (20 μM) under low growth-stimulated conditions. BrdU incorporation in C6 cells (upper panel) and PCNA expression analysis in T98G cells (lower panel) were carried out. Images were captured using a brightfield microscope at a magnification of ×600.

    Techniques Used: Incubation, BrdU Incorporation Assay, Expressing, Microscopy

    Related Articles

    Expressing:

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: Recombinant TNF-α and IFNγ were obtained from Thermo Scientific. .. 15 N-Labeled FAT10 and ubiquitin internal standards were generated by expressing FAT10 or ubiquitin pDEST14 vectors into in Rosetta/DE3 cells (EMD Biosciences, Inc., Darmstadt, Germany) supplemented with BioExpress cell growth media (U-15 N, 98%, Cambridge Isotope Laboratories, Inc., Andover, MA).

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore). .. HASMC were transiently transfected with a plasmid expression vector in which S100A12 is driven by the CMV promoter (in pcDNA3.1, Invitrogen) or vector only (control) using the Neon Transfection System (Invitrogen).

    Positive Control:

    Article Title: Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model
    Article Snippet: .. Recombinant TNF-α (eBioscience) was loaded as a positive control in some experiments. ..

    Quantitative RT-PCR:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. Total RNA was then extracted for the quantification of E-selectin and VCAM-1 mRNAs as well as miR-181b using RT-qPCR.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. Total RNA was then extracted for the quantification of E-selectin and VCAM-1 mRNAs as well as miR-181b using RT-qPCR.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Histone Deacetylase-3 Activation Promotes Tumor Necrosis Factor-? (TNF-?) Expression in Cardiomyocytes during Lipopolysaccharide Stimulation *
    Article Snippet: .. Concentrations of TNF-α protein in culture medium were determined with a mouse TNF-α enzyme-linked immunosorbent assay kit (eBiosciences Inc.) as described in our previous report ( ). .. Chromatin immunoprecipitation assay was performed to assess NF-κB/p65 binding to the TNF-α promoter as described previously ( ).

    Article Title: Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis
    Article Snippet: .. ELISA Assay The protein levels of TNF-α, MCP-1, IL-6 and IL-10 in the supernatants were assessed by ELISA assay with cytokine ELISA kits (eBioscience) according to the manufacturer’s instructions. .. A standard curve was generated using known amounts of the respective purified recombinant mouse cytokines or chemokines.

    Article Title: RelB Modulation of I?B? Stability as a Mechanism of Transcription Suppression of Interleukin-1? (IL-1?), IL-1?, and Tumor Necrosis Factor Alpha in Fibroblasts †
    Article Snippet: .. TNF-α protein was measured with a mouse enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Inc., Camarillo, Calif.). .. Prior to the metabolic labeling, 2 × 106 fibroblasts were treated with 1 μg of LPS per ml for 1 h. The cells were transferred to 1 ml of methionine- and cysteine-free DMEM plus 5% dialyzed FCS for 1 h, and then 0.2 mCi of Tran[35 S] (ICN, Costa Mesa, Calif.) was added to the medium.

    Article Title: Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection
    Article Snippet: TNF-α enzyme-linked immunosorbent assays (ELISAs) were performed with antibody pairs and recombinant TNF-α from eBioscience (San Diego, CA). .. To measure chemokine levels, the following ELISA kits were used: a CCL2 kit from BD Biosciences and CCL7 kit from Bender Med-Systems (Burlingame, CA).

    Article Title: Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells
    Article Snippet: Lipopolysaccharide (LPS), polymyxin B (PmB), and TMB ELISA Substrate were purchased from Sigma–Aldrich (St. Louis, MO). .. Biotinylated monoclonal antibodies directed against human ICAM-1 and VCAM-1 as well as recombinant TNF- α were purchased from e Bioscience (San Diego, CA).

    Article Title: Insights into the pathogenesis of ulcerative colitis from a murine model of stasis-induced dysbiosis, colonic metaplasia, and genetic susceptibility
    Article Snippet: .. ELISA for TNF-α protein on tissue homogenate was performed per manufacturer instructions (catalog no. 88-7324-22, eBiosciences, San Diego, CA). .. Data from the in vivo studies are presented as means ± SE; statistical significance was analyzed by the analysis of variance test (ANOVA) followed by analysis of significance (Tukey-Frame's multiple-comparisons test) or t -test, where appropriate.

    Incubation:

    Article Title: Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model
    Article Snippet: Recombinant TNF-α (eBioscience) was loaded as a positive control in some experiments. .. Membranes were incubated with specific primary antibodies, goat anti-mouse TNF-α antibody (Antigenix America) or rabbit anti-mouse TIMP-3 antibody (Millipore), at 4°C overnight.

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control. .. The membrane was blocked for 90 min using 2% non-fat powder milk (Amersham, EclAdvance™, Buckinghamshire, UK) and was then incubated using the TNF-α antibody (1/1000 dilution, Invitrogen.

    Spectrophotometry:

    Article Title: Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection
    Article Snippet: Cytokine levels were determined by comparison with standard curves generated from recombinant TNF-α (eBioscience). .. Data were analyzed using a Biotek EL808 spectrophotometer.

    Article Title: Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection
    Article Snippet: TNF-α enzyme-linked immunosorbent assays (ELISAs) were performed with antibody pairs and recombinant TNF-α from eBioscience (San Diego, CA). .. Cytokine/chemokine levels were determined by comparison with standard curves, using a Biotek EL808 spectrophotometer.

    Mass Spectrometry:

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: Paragraph title: Mass Spectrometry to Quantitate Intracellular Ubiquitin and FAT10 ... Recombinant TNF-α and IFNγ were obtained from Thermo Scientific.

    Transplantation Assay:

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: Primary cultures of human airway smooth muscle cells (HASMC) were established by the explant method from the trachealis muscle of donors unsuitable for lung transplantation, and with approval form the University of Chicago Institutional Review Board. .. When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore).

    Western Blot:

    Article Title: RelB Modulation of I?B? Stability as a Mechanism of Transcription Suppression of Interleukin-1? (IL-1?), IL-1?, and Tumor Necrosis Factor Alpha in Fibroblasts †
    Article Snippet: Paragraph title: Western blot analysis and ELISA. ... TNF-α protein was measured with a mouse enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Inc., Camarillo, Calif.).

    Article Title: Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model
    Article Snippet: Paragraph title: Western blot ... Recombinant TNF-α (eBioscience) was loaded as a positive control in some experiments.

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: The level of TNF-α in the cerebrospinal fluid was measured using the Western blot test introduced by Boneberg and Hartung. .. To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control.

    Transfection:

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore). .. HASMC were transiently transfected with a plasmid expression vector in which S100A12 is driven by the CMV promoter (in pcDNA3.1, Invitrogen) or vector only (control) using the Neon Transfection System (Invitrogen).

    Activation Assay:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Cell Culture:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: In parallel, HUVECs were specifically isolated for this study and were removed from human umbilical veins by exposure to dispase and cultured in medium 199 (M199, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), antibiotic, antimycotic solution (1%, Sigma), heparin (5 U/mL, Merckle GmbH, Blaubeuren, Germany) and endothelial growth supplement (7.5 ug/mL, Sigma) as described in our previous study [ ]. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: In parallel, HUVECs were specifically isolated for this study and were removed from human umbilical veins by exposure to dispase and cultured in medium 199 (M199, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), antibiotic, antimycotic solution (1%, Sigma), heparin (5 U/mL, Merckle GmbH, Blaubeuren, Germany) and endothelial growth supplement (7.5 ug/mL, Sigma) as described in our previous study [ ]. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: Paragraph title: Cell Culture ... When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore).

    Generated:

    Article Title: Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis
    Article Snippet: ELISA Assay The protein levels of TNF-α, MCP-1, IL-6 and IL-10 in the supernatants were assessed by ELISA assay with cytokine ELISA kits (eBioscience) according to the manufacturer’s instructions. .. A standard curve was generated using known amounts of the respective purified recombinant mouse cytokines or chemokines.

    Article Title: Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection
    Article Snippet: .. Cytokine levels were determined by comparison with standard curves generated from recombinant TNF-α (eBioscience). .. Data were analyzed using a Biotek EL808 spectrophotometer.

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: Cells were harvested and lysates generated using RIPA buffer (Thermo Scientific) supplemented with complete protease inhibitors (Roche Diagnostics) and 10 m m N -ethylmaleimide. .. Recombinant TNF-α and IFNγ were obtained from Thermo Scientific.

    Cytokine Assay:

    Article Title: Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells
    Article Snippet: Biotinylated monoclonal antibodies directed against human ICAM-1 and VCAM-1 as well as recombinant TNF- α were purchased from e Bioscience (San Diego, CA). .. The biotinylated monoclonal antibody directed against E-selectin was purchased from R & D Systems (Minneapolis, MN) as were all cytokine assay kits (Duo-Set ELISA kits) that were used according to the manufacturer’s directions.

    Protein Concentration:

    Article Title: Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model
    Article Snippet: The protein concentration of each supernatant was measured with the Bio-Rad protein assay system (Bio-Rad). .. Recombinant TNF-α (eBioscience) was loaded as a positive control in some experiments.

    Recombinant:

    Article Title: Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis
    Article Snippet: ELISA Assay The protein levels of TNF-α, MCP-1, IL-6 and IL-10 in the supernatants were assessed by ELISA assay with cytokine ELISA kits (eBioscience) according to the manufacturer’s instructions. .. A standard curve was generated using known amounts of the respective purified recombinant mouse cytokines or chemokines.

    Article Title: Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection
    Article Snippet: .. Cytokine levels were determined by comparison with standard curves generated from recombinant TNF-α (eBioscience). .. Data were analyzed using a Biotek EL808 spectrophotometer.

    Article Title: Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection
    Article Snippet: .. TNF-α enzyme-linked immunosorbent assays (ELISAs) were performed with antibody pairs and recombinant TNF-α from eBioscience (San Diego, CA). .. To measure chemokine levels, the following ELISA kits were used: a CCL2 kit from BD Biosciences and CCL7 kit from Bender Med-Systems (Burlingame, CA).

    Article Title: Effect of Stored Product Mite Extracts on Human Dermal Microvascular Endothelial Cells
    Article Snippet: .. Biotinylated monoclonal antibodies directed against human ICAM-1 and VCAM-1 as well as recombinant TNF- α were purchased from e Bioscience (San Diego, CA). .. The biotinylated monoclonal antibody directed against E-selectin was purchased from R & D Systems (Minneapolis, MN) as were all cytokine assay kits (Duo-Set ELISA kits) that were used according to the manufacturer’s directions.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Article Title: Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model
    Article Snippet: .. Recombinant TNF-α (eBioscience) was loaded as a positive control in some experiments. ..

    Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
    Article Snippet: .. After 4 hours medium was changed, and cells were stimulated with recombinant TNF-α (Invitrogen). .. To investigate the relevance of IL-6/gp130-dependent signaling in hepatocytes during chronic cholestatic liver injury we treated wild-type (gp130loxP/loxP ) and hepatocyte-specific gp130 knockout mice (gp130Δhepa ) with a 0.1% DDC-containing diet.

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: .. Recombinant TNF-α and IFNγ were obtained from Thermo Scientific. .. SDS-PAGE was performed using BisTris- and MES-based systems (Invitrogen), and immunoblotting was performed using antibodies to FAT10 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-67203) and Uba6 (Millennium).

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Article Title: Mitochondrial division inhibitor 1 (mdivi-1) enhances death receptor-mediated apoptosis in human ovarian cancer cells
    Article Snippet: .. Recombinant TNF-α was obtained from Invitrogen (Grand Island, NY) and recombinant Super Fas Ligand was obtained from Enzo Life Sciences (Farmingdale, NY). .. The activity of caspase-3/7 was measured using a Caspase-Glo® 3/7 Assay Systems (Promega, Madison, WI), according to the manufacturer's instruction.

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: .. To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control. .. The membrane was blocked for 90 min using 2% non-fat powder milk (Amersham, EclAdvance™, Buckinghamshire, UK) and was then incubated using the TNF-α antibody (1/1000 dilution, Invitrogen.

    Immunofluorescence:

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: SMC were characterized by morphology and immunofluorescence staining using smooth muscle-α-actin antibody (Sigma). .. When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore).

    Isolation:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: In parallel, HUVECs were specifically isolated for this study and were removed from human umbilical veins by exposure to dispase and cultured in medium 199 (M199, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), antibiotic, antimycotic solution (1%, Sigma), heparin (5 U/mL, Merckle GmbH, Blaubeuren, Germany) and endothelial growth supplement (7.5 ug/mL, Sigma) as described in our previous study [ ]. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Article Title: Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis
    Article Snippet: Paragraph title: Primary Hepatocyte Isolation ... After 4 hours medium was changed, and cells were stimulated with recombinant TNF-α (Invitrogen).

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: In parallel, HUVECs were specifically isolated for this study and were removed from human umbilical veins by exposure to dispase and cultured in medium 199 (M199, Gibco, Grand Island, NY, USA) containing 15% fetal bovine serum (Gibco), antibiotic, antimycotic solution (1%, Sigma), heparin (5 U/mL, Merckle GmbH, Blaubeuren, Germany) and endothelial growth supplement (7.5 ug/mL, Sigma) as described in our previous study [ ]. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Purification:

    Article Title: Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis
    Article Snippet: ELISA Assay The protein levels of TNF-α, MCP-1, IL-6 and IL-10 in the supernatants were assessed by ELISA assay with cytokine ELISA kits (eBioscience) according to the manufacturer’s instructions. .. A standard curve was generated using known amounts of the respective purified recombinant mouse cytokines or chemokines.

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: Recombinant TNF-α and IFNγ were obtained from Thermo Scientific. .. Proteins were purified using HiTrap SP column and Superdex 75 column (GE Healthcare).

    Bradford Protein Assay:

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: To ensure the identical protein loading for electrophoresis, the concentration of protein in the cerebrospinal fluid was measured using Bradford protein assay. .. To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control.

    SDS Page:

    Article Title: Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6 *
    Article Snippet: Recombinant TNF-α and IFNγ were obtained from Thermo Scientific. .. SDS-PAGE was performed using BisTris- and MES-based systems (Invitrogen), and immunoblotting was performed using antibodies to FAT10 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-67203) and Uba6 (Millennium).

    Plasmid Preparation:

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore). .. HASMC were transiently transfected with a plasmid expression vector in which S100A12 is driven by the CMV promoter (in pcDNA3.1, Invitrogen) or vector only (control) using the Neon Transfection System (Invitrogen).

    Electrophoresis:

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: A certain amount of the sample under electrophoresis was placed on 12% sodium dodecyl sulfate polyacrylamide gel, and the proteins were then transferred to the polyvinylidene difluoride membrane. .. To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control.

    In Vitro:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation. .. In parallel, the effect of everolimus on EC activation was studied using everolimus (0.5 μM, dissolved in DMSO, Sigma) in the presence of TNF-α for the same time period above.

    Quantitation Assay:

    Article Title: Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection
    Article Snippet: TNF-α enzyme-linked immunosorbent assays (ELISAs) were performed with antibody pairs and recombinant TNF-α from eBioscience (San Diego, CA). .. NO• concentrations were determined with a Nitric Oxide Quantitation Kit (Active Motif, Carlsbad, CA).

    Subculturing Assay:

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: For subculturing, cell density was set to 5,000 cells per cm2 in both cell cultures. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Article Title: Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting
    Article Snippet: For subculturing, cell density was set to 5,000 cells per cm2 in both cell cultures. .. HCAEC and HUVEC cells (3x105 /well) were treated in 6-well plates with recombinant TNF-α (100 ng/mL, Gibco) for 1–24 hours to generate cellular inflammatory conditions as an in vitro model of stent-induced EC inflammation.

    Produced:

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control. .. The membrane was incubated using horseradish peroxidase (HRP) -conjugated secondary antibodies, and the X-ray film was produced within 1 min using chemiluminescence kit.

    Concentration Assay:

    Article Title: Forced exercise attenuates neuropathic pain in chronic constriction injury of male rat: an investigation of oxidative stress and inflammation
    Article Snippet: To ensure the identical protein loading for electrophoresis, the concentration of protein in the cerebrospinal fluid was measured using Bradford protein assay. .. To quantify the TNF-α level of cerebrospinal fluid, 4 ng of recombinant TNF-α (eBioscience, Vienna, Austria) was loaded on the gel as a standard control.

    Staining:

    Article Title: Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation
    Article Snippet: SMC were characterized by morphology and immunofluorescence staining using smooth muscle-α-actin antibody (Sigma). .. When indicated, cells were stimulated with recombinant TNF-α (0.1 ng/ml) and IFN-γ (10 ng/ml, Pierce), or with activating Fas monoclonal antibody (clone CH11, 50 ng/ml, Millipore).

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