aa98  (Millipore)


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  • 89
    Name:
    Anti Mouse Polyvalent Immunoglobulins antibody
    Description:

    Catalog Number:
    m6149
    Price:
    None
    Applications:
    Anti-Mouse Polyvalent Immunoglobulins antibody produced in goat was used to determine immunoglobulin levels in the blood of SCID mouse by ELISA.
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    Structured Review

    Millipore aa98
    CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody <t>AA98</t> was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P

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    aa98 - by Bioz Stars, 2020-09
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    1) Product Images from "CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development"

    Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

    Journal: Cell Research

    doi: 10.1038/cr.2015.15

    CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody AA98 was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P
    Figure Legend Snippet: CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody AA98 was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P

    Techniques Used: Mouse Assay, Aortic Ring Assay, Matrigel Assay, Injection

    2) Product Images from "Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound"

    Article Title: Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2013.05.008

    MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p
    Figure Legend Snippet: MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p

    Techniques Used: Immunohistochemistry

    MRIgFUS facilitates the entry of IgG and IgM into the brain. Using immunohistochemistry, high levels of IgG were detected in MRIgFUS-treated compared to untreated cortex of TgCRND8 ( A ) and non-Tg ( B ) mice. Similarly, IgM observed in MRIgFUS-treated cortical regions was greater than that detected in contralateral cortex of TgCRND8 ( C ) and non-Tg mice ( D ). Quantitative western blot analysis revealed that compared to untreated cortex, the cortex treated with MRIgFUS had greater levels of IgG in TgCRND8 ( E ) and non-Tg ( F ) mice; and of IgM in TgCRND8 ( G ) and non-Tg ( H ) mice. Scale bar: A–D= 500 μm. Data are presented as the mean ± SEM, *p
    Figure Legend Snippet: MRIgFUS facilitates the entry of IgG and IgM into the brain. Using immunohistochemistry, high levels of IgG were detected in MRIgFUS-treated compared to untreated cortex of TgCRND8 ( A ) and non-Tg ( B ) mice. Similarly, IgM observed in MRIgFUS-treated cortical regions was greater than that detected in contralateral cortex of TgCRND8 ( C ) and non-Tg mice ( D ). Quantitative western blot analysis revealed that compared to untreated cortex, the cortex treated with MRIgFUS had greater levels of IgG in TgCRND8 ( E ) and non-Tg ( F ) mice; and of IgM in TgCRND8 ( G ) and non-Tg ( H ) mice. Scale bar: A–D= 500 μm. Data are presented as the mean ± SEM, *p

    Techniques Used: Immunohistochemistry, Mouse Assay, Western Blot

    MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p
    Figure Legend Snippet: MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p

    Techniques Used: Immunohistochemistry

    3) Product Images from "Semen activates the female immune response during early pregnancy in mice"

    Article Title: Semen activates the female immune response during early pregnancy in mice

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2004.01876.x

    Seminal fluid is necessary for lymphocyte activation in PALN following mating. The effect of sperm and seminal vesicle fluid on the immune response elicited in the PALN following mating was evaluated using surgically altered male mice. Females were mated with intact BALB/c males or with BALB/c males rendered sperm deficient by vasectomy (Vas) or seminal vesicle deficient (SV – ) by surgical removal of the seminal vesicles. (a) The mean ± SEM number of cells retrieved from PALN. (b) The percentage of cytokine-producing cells in CD3 + lymphocytes from PALN of day 4 mated mice was determined by flow cytometry using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4- and IFN-γ-positive cells. Data were compared by independent samples t -test. The number of mice per group are shown in parentheses. * P
    Figure Legend Snippet: Seminal fluid is necessary for lymphocyte activation in PALN following mating. The effect of sperm and seminal vesicle fluid on the immune response elicited in the PALN following mating was evaluated using surgically altered male mice. Females were mated with intact BALB/c males or with BALB/c males rendered sperm deficient by vasectomy (Vas) or seminal vesicle deficient (SV – ) by surgical removal of the seminal vesicles. (a) The mean ± SEM number of cells retrieved from PALN. (b) The percentage of cytokine-producing cells in CD3 + lymphocytes from PALN of day 4 mated mice was determined by flow cytometry using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4- and IFN-γ-positive cells. Data were compared by independent samples t -test. The number of mice per group are shown in parentheses. * P

    Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry

    Cytokine synthesis assessed by immunohistochemistry is increased in PALN lymphocytes after insemination. Frozen sections of PALN tissue taken from virgin or day 4 mated mice were labelled with rat anti-IFN-γ or anti-IL-4 antibodies followed by biotin-conjugated rabbit anti-rat IgG. (a) IFN-γ-producing cells were sparsely scattered within the PALN of virgin mice, and (b) were considerably increased in abundance following mating. (c) IL-5-producing cells were rare in the PALN of virgin mice, and (d) were increased following mating. Staining was absent in tissues from both virgin (e) and day 4 mated mice (f) probed with an isotype-matched control antibody. Scale bar = 100 μm. (g) Stained cells were quantified by manual counting and are expressed as the mean ± SEM number of positive cells per section in virgin and day 4 mated PALN sections. One to three PALN sections from n = 10 virgin and n = 8 day 4 mated mice were examined. * P = 0·02.
    Figure Legend Snippet: Cytokine synthesis assessed by immunohistochemistry is increased in PALN lymphocytes after insemination. Frozen sections of PALN tissue taken from virgin or day 4 mated mice were labelled with rat anti-IFN-γ or anti-IL-4 antibodies followed by biotin-conjugated rabbit anti-rat IgG. (a) IFN-γ-producing cells were sparsely scattered within the PALN of virgin mice, and (b) were considerably increased in abundance following mating. (c) IL-5-producing cells were rare in the PALN of virgin mice, and (d) were increased following mating. Staining was absent in tissues from both virgin (e) and day 4 mated mice (f) probed with an isotype-matched control antibody. Scale bar = 100 μm. (g) Stained cells were quantified by manual counting and are expressed as the mean ± SEM number of positive cells per section in virgin and day 4 mated PALN sections. One to three PALN sections from n = 10 virgin and n = 8 day 4 mated mice were examined. * P = 0·02.

    Techniques Used: Immunohistochemistry, Mouse Assay, Staining

    Cytokine synthesis assessed by intracellular FACS is increased in PALN T lymphocytes after insemination. (a) PALN cells from virgin mice (left hand panel) and day 4 mated mice (right hand panel) were stimulated for 6 hr with PMA and calcium ionophore in the presence of monensin, then stained for intracellular IL-4, IL-5, or IFN-γ. The data shown are representative of at least 10 independent experiments. (b) The percentage of cytokine-producing cells in CD3 + lymphocytes from PALN of day 4 mated mice was determined using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4-, IL-5- and IFN-γ-positive cells. PALN from n = 25 virgin and n = 24 day 4 mated mice were assessed. Data were compared by independent samples t -test. * P = 0·04; ** P = 0·003.
    Figure Legend Snippet: Cytokine synthesis assessed by intracellular FACS is increased in PALN T lymphocytes after insemination. (a) PALN cells from virgin mice (left hand panel) and day 4 mated mice (right hand panel) were stimulated for 6 hr with PMA and calcium ionophore in the presence of monensin, then stained for intracellular IL-4, IL-5, or IFN-γ. The data shown are representative of at least 10 independent experiments. (b) The percentage of cytokine-producing cells in CD3 + lymphocytes from PALN of day 4 mated mice was determined using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4-, IL-5- and IFN-γ-positive cells. PALN from n = 25 virgin and n = 24 day 4 mated mice were assessed. Data were compared by independent samples t -test. * P = 0·04; ** P = 0·003.

    Techniques Used: FACS, Mouse Assay, Staining

    4) Product Images from "Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation ▿-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation ▿ †"

    Article Title: Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation ▿-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.02047-06

    Hyperresponsive phenotypes of Cmah -null mice. (A) T-independent hyperresponse of Cmah -null mice. DNP-Ficoll was used to immunize 8-week-old mice. Serum was collected each week and analyzed for reactivity with DNP-conjugated BSA coated on ELISA plates. The titer of hapten-reacting mouse Igs from each animal was determined by isotype-specific ELISA. The measured optical density at 405 nm was normalized to anti-DNP units by comparison with the value from standard pooled serum against DNP on the same plate. The results are presented as the mean responses of 10 animals for each genotype measured in two sets of experiments. The bars represent standard errors of the means. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah -null mice for each isotype. Genotypes are indicated as follows: +/+, wild-type; −/−, Cmah -null (A and B). (B) Normal T-dependent immune response of Cmah -null mice. DNP-KLH in complete Freund's adjuvant was used to immunize 8-week-old mice. The titers of hapten-reacting mouse Igs from each animal were determined by isotype-specific ELISA as above. Arrows indicate the time of secondary immunization with DNP-KLH. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah -null mice for each isotype. (C) In vitro hyperproliferation response of Cmah -null B cells. Splenic B cells from wild-type (open columns) and Cmah -null (filled columns) mice were assessed for proliferation using the F(ab′) 2 fragment of anti-mouse IgM (μ chain) or anti-IgM plus 2 ng/ml IL-4 as stimulating reagents. After stimulation for 24 h, BrdU was added. Following incubation overnight, incorporated BrdU was detected by ELISA. Data are shown as the means of triplicate cultures, and the bars represent standard errors of the means. The results shown here were obtained in one of the experiments using 10% FBS-containing medium.
    Figure Legend Snippet: Hyperresponsive phenotypes of Cmah -null mice. (A) T-independent hyperresponse of Cmah -null mice. DNP-Ficoll was used to immunize 8-week-old mice. Serum was collected each week and analyzed for reactivity with DNP-conjugated BSA coated on ELISA plates. The titer of hapten-reacting mouse Igs from each animal was determined by isotype-specific ELISA. The measured optical density at 405 nm was normalized to anti-DNP units by comparison with the value from standard pooled serum against DNP on the same plate. The results are presented as the mean responses of 10 animals for each genotype measured in two sets of experiments. The bars represent standard errors of the means. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah -null mice for each isotype. Genotypes are indicated as follows: +/+, wild-type; −/−, Cmah -null (A and B). (B) Normal T-dependent immune response of Cmah -null mice. DNP-KLH in complete Freund's adjuvant was used to immunize 8-week-old mice. The titers of hapten-reacting mouse Igs from each animal were determined by isotype-specific ELISA as above. Arrows indicate the time of secondary immunization with DNP-KLH. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah -null mice for each isotype. (C) In vitro hyperproliferation response of Cmah -null B cells. Splenic B cells from wild-type (open columns) and Cmah -null (filled columns) mice were assessed for proliferation using the F(ab′) 2 fragment of anti-mouse IgM (μ chain) or anti-IgM plus 2 ng/ml IL-4 as stimulating reagents. After stimulation for 24 h, BrdU was added. Following incubation overnight, incorporated BrdU was detected by ELISA. Data are shown as the means of triplicate cultures, and the bars represent standard errors of the means. The results shown here were obtained in one of the experiments using 10% FBS-containing medium.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Incubation

    5) Product Images from "Evolutionary conservation of neuropeptide expression in the thymus of different species"

    Article Title: Evolutionary conservation of neuropeptide expression in the thymus of different species

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2006.02351.x

    Immunoperoxidase staining for neuropeptides in thymus of 10-week-old mouse. Frozen mouse thymic sections were stained with anti-CGRP (a), anti-NPY (b), anti-SOM (c), anti-SP (d) and anti-VIP (e) antibodies. Immunoreactive cells are marked by arrows both
    Figure Legend Snippet: Immunoperoxidase staining for neuropeptides in thymus of 10-week-old mouse. Frozen mouse thymic sections were stained with anti-CGRP (a), anti-NPY (b), anti-SOM (c), anti-SP (d) and anti-VIP (e) antibodies. Immunoreactive cells are marked by arrows both

    Techniques Used: Immunoperoxidase Staining, Staining

    RT-PCR analysis of neuropeptide mRNA expression in the thymus and brain from mouse, chicken and Xenopus . In the mouse thymus, mRNA for CGRP (lane 2, 320 bp), NPY (lane 3, 288 bp), SOM (lane 4, 352 bp), SP (lane 5, 215 bp) and VIP (lane 6, 245 bp) were
    Figure Legend Snippet: RT-PCR analysis of neuropeptide mRNA expression in the thymus and brain from mouse, chicken and Xenopus . In the mouse thymus, mRNA for CGRP (lane 2, 320 bp), NPY (lane 3, 288 bp), SOM (lane 4, 352 bp), SP (lane 5, 215 bp) and VIP (lane 6, 245 bp) were

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    6) Product Images from "A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway "

    Article Title: A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway

    Journal: The Journal of Cell Biology

    doi:

    Expression of T7Hub increases steady-state levels of cotransfected Tac chimeras bearing tyrosine- and dileucine-based sorting motifs and causes their cell surface accumulation. HeLa cells were transfected with control vector without the hub insert ( V ) or T7Hub ( H ). Each population of cells was cotransfected with vector encoding the Tac chimera with the tyrosine-based sorting signal of H2M ( Y ) or vector encoding the Tac chimera with the dileucine-based sorting motif of CD3γ ( LL ). 48 h after transfection, cells were surface biotinylated and lysates were prepared. Surface molecules were removed from the lysate with avidin–agarose and then internal molecules were left in the unbound fraction. The presence of Tac chimeras in each fraction of the lysate was established by immunoblotting with anti-Tac antiserum after SDS-PAGE. For each sample, 100% of the surface fraction and 25% of the internal fraction were analyzed.
    Figure Legend Snippet: Expression of T7Hub increases steady-state levels of cotransfected Tac chimeras bearing tyrosine- and dileucine-based sorting motifs and causes their cell surface accumulation. HeLa cells were transfected with control vector without the hub insert ( V ) or T7Hub ( H ). Each population of cells was cotransfected with vector encoding the Tac chimera with the tyrosine-based sorting signal of H2M ( Y ) or vector encoding the Tac chimera with the dileucine-based sorting motif of CD3γ ( LL ). 48 h after transfection, cells were surface biotinylated and lysates were prepared. Surface molecules were removed from the lysate with avidin–agarose and then internal molecules were left in the unbound fraction. The presence of Tac chimeras in each fraction of the lysate was established by immunoblotting with anti-Tac antiserum after SDS-PAGE. For each sample, 100% of the surface fraction and 25% of the internal fraction were analyzed.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Avidin-Biotin Assay, SDS Page

    Expression of T7Hub in transfected cells does not alter the general distribution of adaptor molecules. HeLa cells were transfected with T7Hub and after 48 h were then analyzed by immunofluorescence for the distribution of the AP1 and AP2 adaptors. ( A ) Cells stained with anti-AP2 mAb AP.6 ( AP2 ) and biotinylated anti-T7 mAb followed by FITC-conjugated anti–mouse IgG 1 –specific secondary antibody (for AP.6) and TRITC-conjugated streptavidin. ( B ) Cells stained with anti-AP1 mAb 100/3 ( AP1 ) and anti-LC ( anti-LC ) antiserum followed by LRSC-conjugated goat anti–mouse IgG and FITC-conjugated goat anti– rabbit IgG. The T7Hub– transfected cells in B were identified by the loss of punctate clathrin LC staining ( arrowheads ).
    Figure Legend Snippet: Expression of T7Hub in transfected cells does not alter the general distribution of adaptor molecules. HeLa cells were transfected with T7Hub and after 48 h were then analyzed by immunofluorescence for the distribution of the AP1 and AP2 adaptors. ( A ) Cells stained with anti-AP2 mAb AP.6 ( AP2 ) and biotinylated anti-T7 mAb followed by FITC-conjugated anti–mouse IgG 1 –specific secondary antibody (for AP.6) and TRITC-conjugated streptavidin. ( B ) Cells stained with anti-AP1 mAb 100/3 ( AP1 ) and anti-LC ( anti-LC ) antiserum followed by LRSC-conjugated goat anti–mouse IgG and FITC-conjugated goat anti– rabbit IgG. The T7Hub– transfected cells in B were identified by the loss of punctate clathrin LC staining ( arrowheads ).

    Techniques Used: Expressing, Transfection, Immunofluorescence, Staining

    Hub expression does not have pleiotropic effects on organelle integrity or the constitutive secretory pathway. HeLa cells were transfected with T7Hub and, after 48 h, were stained with ( A ) anti-ER antiserum and FITC-conjugated goat anti– rabbit IgG, ( B ) anti–β-COP mAb M3A5 and FITC-conjugated goat anti–mouse IgG 1 –specific secondary antibody, or ( C ) anti-MHC class I mAb W6/32 and FITC-conjugated anti–mouse IgG 2a –specific secondary antibody. The T7Hub-transfected cells in each field were visualized either by ( A ) anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG or ( B and C ) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.
    Figure Legend Snippet: Hub expression does not have pleiotropic effects on organelle integrity or the constitutive secretory pathway. HeLa cells were transfected with T7Hub and, after 48 h, were stained with ( A ) anti-ER antiserum and FITC-conjugated goat anti– rabbit IgG, ( B ) anti–β-COP mAb M3A5 and FITC-conjugated goat anti–mouse IgG 1 –specific secondary antibody, or ( C ) anti-MHC class I mAb W6/32 and FITC-conjugated anti–mouse IgG 2a –specific secondary antibody. The T7Hub-transfected cells in each field were visualized either by ( A ) anti-T7 mAb followed by LRSC-conjugated goat anti–mouse IgG or ( B and C ) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.

    Techniques Used: Expressing, Transfection, Staining

    Direct delivery of I chain (coexpressed with DR) from the TGN to the endocytic pathway is not affected by expression of hubs. HeLa cells were transfected with ( A ) control vector (no hub) or ( B and C ) T7Hub and ( A–C ) cotransfected with vectors encoding HLA-DR α and β chains (pCDM8DRα and pCDM8DRβ) and the I chain driven by human metallothionein promoter (pMTΔ302Ip33). 24 h after transfection, I chain expression was induced by exposure to 10 μM CdCl 2 for 20 h. Cells were fixed, permeabilized, and then processed for immunofluorescence. The cells were stained with ( A and B ) anti-I chain mAb PIN.1.1, followed by FITC-conjugated goat anti–mouse IgG 1 – specific secondary antibody and then ( C ) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.
    Figure Legend Snippet: Direct delivery of I chain (coexpressed with DR) from the TGN to the endocytic pathway is not affected by expression of hubs. HeLa cells were transfected with ( A ) control vector (no hub) or ( B and C ) T7Hub and ( A–C ) cotransfected with vectors encoding HLA-DR α and β chains (pCDM8DRα and pCDM8DRβ) and the I chain driven by human metallothionein promoter (pMTΔ302Ip33). 24 h after transfection, I chain expression was induced by exposure to 10 μM CdCl 2 for 20 h. Cells were fixed, permeabilized, and then processed for immunofluorescence. The cells were stained with ( A and B ) anti-I chain mAb PIN.1.1, followed by FITC-conjugated goat anti–mouse IgG 1 – specific secondary antibody and then ( C ) biotinylated anti-T7 mAb followed by TRITC-conjugated streptavidin.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Staining

    7) Product Images from "Attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice"

    Article Title: Attenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice

    Journal: Brain Research

    doi: 10.1016/j.brainres.2009.06.007

    Minocycline has anti-inflammatory effects in ts 1-infected mice brainstem and cultured C1 astrocytes. (A). Cell lysates or nuclear extracts from uninfected, ts 1-only and ts 1-mino C1 astrocytes are probed with anti-lκBα, NF-κB p65 and COX-2. (B). Mice from two groups were sacrificed at 30 dpi. Paraffin sections of the brainstem used for immunohistochemistry (IHC) assay. These sections were incubated with anti-GFAP (brown) and anti-COX-2 antibodies as specified in Experimental Procedures. Strong COX-2 immunoreactivity (red) is evident in astrocyte (arrow) of ts 1-only mouse (left panel), but weak in ts 1-mino mouse (right panel). Magnifications: 40X. (C). Expression of CD68, which marks activated microglia and macrophages, was examined by immunofluorescence staining using brainstem sections from uninfected, ts 1-only and ts 1-mino mice. Magnifications: 20X.
    Figure Legend Snippet: Minocycline has anti-inflammatory effects in ts 1-infected mice brainstem and cultured C1 astrocytes. (A). Cell lysates or nuclear extracts from uninfected, ts 1-only and ts 1-mino C1 astrocytes are probed with anti-lκBα, NF-κB p65 and COX-2. (B). Mice from two groups were sacrificed at 30 dpi. Paraffin sections of the brainstem used for immunohistochemistry (IHC) assay. These sections were incubated with anti-GFAP (brown) and anti-COX-2 antibodies as specified in Experimental Procedures. Strong COX-2 immunoreactivity (red) is evident in astrocyte (arrow) of ts 1-only mouse (left panel), but weak in ts 1-mino mouse (right panel). Magnifications: 40X. (C). Expression of CD68, which marks activated microglia and macrophages, was examined by immunofluorescence staining using brainstem sections from uninfected, ts 1-only and ts 1-mino mice. Magnifications: 20X.

    Techniques Used: Infection, Mouse Assay, Cell Culture, Immunohistochemistry, Incubation, Expressing, Immunofluorescence, Staining

    8) Product Images from "Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets"

    Article Title: Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets

    Journal: Infection and Immunity

    doi:

    Inhibition of biotinylated S. aureus Cowan I and Wood 46 strain (1% cell suspension) adhesion to immobilized, glutaraldehyde-fixed platelets by soluble protein A (0.25 mg/ml). Bacterial cell adhesion was evaluated after 60 min at 37°C, using AP-STRAV and pNPP substrate. The data summarize bacterial cell binding to platelets in the presence of protein A relative to binding in the absence of protein A. Values represent means ± SD; n = 3.
    Figure Legend Snippet: Inhibition of biotinylated S. aureus Cowan I and Wood 46 strain (1% cell suspension) adhesion to immobilized, glutaraldehyde-fixed platelets by soluble protein A (0.25 mg/ml). Bacterial cell adhesion was evaluated after 60 min at 37°C, using AP-STRAV and pNPP substrate. The data summarize bacterial cell binding to platelets in the presence of protein A relative to binding in the absence of protein A. Values represent means ± SD; n = 3.

    Techniques Used: Inhibition, Binding Assay

    9) Product Images from "Characterisation of the bovine enteric calici-like virus, Newbury agent 1"

    Article Title: Characterisation of the bovine enteric calici-like virus, Newbury agent 1

    Journal: FEMS Microbiology Letters

    doi: 10.1016/S0378-1097(00)00422-5

    (a) Molecular mass estimation of the capsid protein of NA1 particles purified by CsCl density gradient centrifugation. M, molecular masses of biotinylated standard proteins (kDa). Track 1: bovine IgG as a control for blotting and staining with the anti-bovine conjugate; track 2: biotinylated standard proteins; track 3: biotinylated standard proteins plus the NA1 capsid protein; track 4: the NA1 capsid protein; arrow indicates the NA1 protein. (b) Molecular mass estimation of the capsid protein of FCV grown in CRFK cells using the same electrophoresis conditions as those used for NA1. Arrow indicates the FCV capsid protein. M, molecular masses of biotinylated standard proteins (kDa). Track 1: preparation from uninfected CRFK cells; track 2: biotinylated standard proteins plus the preparation from FCV-infected cells; track 3: FCV-infected cells; track 4: rabbit IgG (the FCV NADC rabbit antiserum) used as a control for blotting and immunostaining with the anti-rabbit conjugate.
    Figure Legend Snippet: (a) Molecular mass estimation of the capsid protein of NA1 particles purified by CsCl density gradient centrifugation. M, molecular masses of biotinylated standard proteins (kDa). Track 1: bovine IgG as a control for blotting and staining with the anti-bovine conjugate; track 2: biotinylated standard proteins; track 3: biotinylated standard proteins plus the NA1 capsid protein; track 4: the NA1 capsid protein; arrow indicates the NA1 protein. (b) Molecular mass estimation of the capsid protein of FCV grown in CRFK cells using the same electrophoresis conditions as those used for NA1. Arrow indicates the FCV capsid protein. M, molecular masses of biotinylated standard proteins (kDa). Track 1: preparation from uninfected CRFK cells; track 2: biotinylated standard proteins plus the preparation from FCV-infected cells; track 3: FCV-infected cells; track 4: rabbit IgG (the FCV NADC rabbit antiserum) used as a control for blotting and immunostaining with the anti-rabbit conjugate.

    Techniques Used: Purification, Gradient Centrifugation, Staining, Electrophoresis, Infection, Immunostaining

    10) Product Images from "Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury"

    Article Title: Propolis Modulates Fibronectin Expression in the Matrix of Thermal Injury

    Journal: BioMed Research International

    doi: 10.1155/2014/748101

    The characteristics of molecular profile of extracts differently treated burn wounds buffered by urea solution and sodium dodecyl sulfate (SDS). Extract components, subjected to dithiothreitol as a factor reducing the disulfide bonds, were subjected to the electrophoresis in a 6% polyacrylamide gel in the presence of SDS. Lane 1, molecular mass markers; lane 2, standard of collagen type I; lanes 3, 4, 5, and 6, components extracted, on the 5th day of healing, from wounds treated by NaCl, SSD, propolis, and propolis vehicle, respectively. The arrows indicate the migration position of standards of known molecular weights (60 kDa, 100 kDa, and 220 kDa).
    Figure Legend Snippet: The characteristics of molecular profile of extracts differently treated burn wounds buffered by urea solution and sodium dodecyl sulfate (SDS). Extract components, subjected to dithiothreitol as a factor reducing the disulfide bonds, were subjected to the electrophoresis in a 6% polyacrylamide gel in the presence of SDS. Lane 1, molecular mass markers; lane 2, standard of collagen type I; lanes 3, 4, 5, and 6, components extracted, on the 5th day of healing, from wounds treated by NaCl, SSD, propolis, and propolis vehicle, respectively. The arrows indicate the migration position of standards of known molecular weights (60 kDa, 100 kDa, and 220 kDa).

    Techniques Used: Electrophoresis, Migration

    11) Product Images from "Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type"

    Article Title: Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-11-333

    Nucant pseudopeptides are internalized into cells and accumulate in the cytoplasm without translocation to the nucleus . A/B. HuT 78 cells in polylysine-coated eight-well glass slides were incubated (37°C) with 15 μM of biotinylated N3 or N7 for 5 hours before PFA/Triton fixation and processing for immunofluorescence microscopy using rabbit anti-biotin and FITC conjugated goat anti-rabbit IgG. C. HuT 78 cells in polylysine-coated slides were incubated (37°C) with 15 μM of biotinylated N7 for 5 hours before methanol/acetate fixation and processing for immunofluorescence microscopy as in A/B (Methods). The nuclei stained with DAPI are shown on the right of each panel. Treatment of HuT 78 cells at 15 μM of biotinylated N3 or N7 leads to 25 and 40% cell death after 24 of culture, respectively.
    Figure Legend Snippet: Nucant pseudopeptides are internalized into cells and accumulate in the cytoplasm without translocation to the nucleus . A/B. HuT 78 cells in polylysine-coated eight-well glass slides were incubated (37°C) with 15 μM of biotinylated N3 or N7 for 5 hours before PFA/Triton fixation and processing for immunofluorescence microscopy using rabbit anti-biotin and FITC conjugated goat anti-rabbit IgG. C. HuT 78 cells in polylysine-coated slides were incubated (37°C) with 15 μM of biotinylated N7 for 5 hours before methanol/acetate fixation and processing for immunofluorescence microscopy as in A/B (Methods). The nuclei stained with DAPI are shown on the right of each panel. Treatment of HuT 78 cells at 15 μM of biotinylated N3 or N7 leads to 25 and 40% cell death after 24 of culture, respectively.

    Techniques Used: Translocation Assay, Incubation, Immunofluorescence, Microscopy, Staining

    12) Product Images from "Targeted Disruption of Fibronectin-Integrin Interactions in Human Gingival Fibroblasts by the RI Protease of Porphyromonas gingivalis W50"

    Article Title: Targeted Disruption of Fibronectin-Integrin Interactions in Human Gingival Fibroblasts by the RI Protease of Porphyromonas gingivalis W50

    Journal: Infection and Immunity

    doi:

    HGF incubated for 1 h with TLCK-treated RI (A to I) or TLCK-treated RIA (J to L). The first column shows cells stained with the PgWC antibody, and the second column shows the same cells double labelled with mouse antibodies against β 1 integrin (B), α V integrin (E), and fibronectin (H and K). The final column shows cells multiply exposed to both labels. RI-treated cells showed a comparable linear distribution of both P. gingivalis components and β 1 integrin along the cytoplasm (C), whereas the peripheral distribution of α V integrin mainly around the tips of the cell differed from the more central staining with PgWC (F). Exact colocalization of fibronectin and P. gingivalis components in RI-treated cells is indicated by the yellow, generally linear, cytoplasmic staining (I), whereas the weaker staining of P. gingivalis components in RIA-treated cells resulted in a predominantly green staining due to the fibronectin (L). Identical exposure periods were used for panels I and L. The patterns of staining with α 5 and β 3 integrin subunits (not shown) resembled those illustrated for β 1 and α V respectively. Magnification, ×750; bar = 10 μm.
    Figure Legend Snippet: HGF incubated for 1 h with TLCK-treated RI (A to I) or TLCK-treated RIA (J to L). The first column shows cells stained with the PgWC antibody, and the second column shows the same cells double labelled with mouse antibodies against β 1 integrin (B), α V integrin (E), and fibronectin (H and K). The final column shows cells multiply exposed to both labels. RI-treated cells showed a comparable linear distribution of both P. gingivalis components and β 1 integrin along the cytoplasm (C), whereas the peripheral distribution of α V integrin mainly around the tips of the cell differed from the more central staining with PgWC (F). Exact colocalization of fibronectin and P. gingivalis components in RI-treated cells is indicated by the yellow, generally linear, cytoplasmic staining (I), whereas the weaker staining of P. gingivalis components in RIA-treated cells resulted in a predominantly green staining due to the fibronectin (L). Identical exposure periods were used for panels I and L. The patterns of staining with α 5 and β 3 integrin subunits (not shown) resembled those illustrated for β 1 and α V respectively. Magnification, ×750; bar = 10 μm.

    Techniques Used: Incubation, Staining

    HGF stained to demonstrate fibronectin after incubation for 1 h with RI (A) or RIA (B), both at 0.2 U of arginine-specific enzyme activity per ml, compared with control cells in DMEM (C). RI-treated cells show only remnants of fine granular cytoplasmic staining and often an indistinct nucleus, in contrast with the clear fibronectin network associated with RIA-treated and control cells. Following removal of cells, the fibronectin matrix was totally disrupted after exposure to RI at 0.2 U/ml (D), whereas a clear fibronectin network was retained in the presence of a similar concentration of RIA (E) and in the DMEM control (F). Magnification, ×750; bar = 10 μm.
    Figure Legend Snippet: HGF stained to demonstrate fibronectin after incubation for 1 h with RI (A) or RIA (B), both at 0.2 U of arginine-specific enzyme activity per ml, compared with control cells in DMEM (C). RI-treated cells show only remnants of fine granular cytoplasmic staining and often an indistinct nucleus, in contrast with the clear fibronectin network associated with RIA-treated and control cells. Following removal of cells, the fibronectin matrix was totally disrupted after exposure to RI at 0.2 U/ml (D), whereas a clear fibronectin network was retained in the presence of a similar concentration of RIA (E) and in the DMEM control (F). Magnification, ×750; bar = 10 μm.

    Techniques Used: Staining, Incubation, Activity Assay, Concentration Assay

    13) Product Images from "Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo"

    Article Title: Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2011.4

    Shigella -infected HeLa cells undergo intrinsic apoptosis. ( a ) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. ( b ) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In ( a and b ), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H 2 O 2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with si RNA for caspase-9 ( c ) or for caspase-8 ( d ) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF- α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. * P
    Figure Legend Snippet: Shigella -infected HeLa cells undergo intrinsic apoptosis. ( a ) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. ( b ) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In ( a and b ), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H 2 O 2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with si RNA for caspase-9 ( c ) or for caspase-8 ( d ) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF- α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. * P

    Techniques Used: Infection, FACS, Staining, Activation Assay, Labeling, Transfection, Activity Assay

    Apoptosis assessment and Gadd45 α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. ( A ) Dot blot of the distribution of LPS, Gadd45 α , TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. *** P
    Figure Legend Snippet: Apoptosis assessment and Gadd45 α expression in a human ex vivo organ culture (EVOC) invasion assay with S. flexneri M90T. ( A ) Dot blot of the distribution of LPS, Gadd45 α , TUNEL and caspase-3 immunostained epithelial cells in sections of human colon biopsies infected with M90T or BS176 (12 h) or uninfected. Immunohistochemically stained cells were counted at × 400 magnification. For each sample, five view fields in five sections were considered for cell enumeration. Circles represent the mean values per sample while the horizontal line represents the mean value per sample category. *** P

    Techniques Used: Expressing, Ex Vivo, Organ Culture, Invasion Assay, Dot Blot, TUNEL Assay, Infection, Staining

    14) Product Images from "Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells"

    Article Title: Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M033639

    Reconstitution of lipid-regulated ERAD of mammalian Insig-1 in Drosophila S2 cells. S2 cells were set up in 6-well plates on day 0 at 1 × 10 6 cells per well in medium A supplemented with 10% HI-FCS. On day 1, cells were transfected in medium B using Maxfect™ as follows. A: 0.1 µg of pAc-Insig-1-myc or 0.1 µg of pAc-Insig-2-myc. B: 0.2 µg pAc-Insig-1-Myc in the absence or presence of 1.0 µg pAc-HA-ubiquitin. C: 0.1 µg of pAc-Insig-1-myc and 1, 3, 10, or 30 ng of pAc-Scap. D: 0.1 of µg pAc-Insig-1-myc. Total amount of DNA was adjusted to 0.1 μg (A, D), 1.2 μg (B), or 0.13 μg (C) using empty pAc5.1 vector. On day 2, each well received 1 ml of medium B supplemented with 20% HI-LPDS (A–C) or HI-DFCS (D). Cells were treated on day 3 with medium C supplemented with 10% HI-LPDS (A–C) or HI-DFCS (D) under the following conditions. A: In the absence or presence of 10 μM MG-132 (6 h) and 50 μM cycloheximide (2 h). B: In the presence of 10 µM MG-132 (2 h). C: In the absence and presence of 2.5 μM 25-HC and 10 mM mevalonate (4 h) together with 50 μM cycloheximide (2 h). D: In the absence and presence of 10 μM MG-132 (6 h) or 0.1, 0.3, or 1 mM BSA-oleate (4 h) together with 50 μM cycloheximide (2 h). A, C, D: Following incubations, cells were harvested and aliquots of whole cell lysates [30 μg protein/lane (A), 40 μg protein/lane (C), 50 μg protein/lane (D)] were subjected to 10% SDS-PAGE followed by immunoblot analysis with IgG-9E10 (against Insigs), IgG-9D5 (against hamster Scap), and anti-actin IgG. B: Following incubation, the cells were harvested for preparation of detergent lysates that were immunoprecipitated with 60 µl anti-Myc coupled agarose beads. Aliquots of the immunoprecipitates were then subjected to SDS-PAGE followed by immunoblot analysis with anti-HA (against ubiquitin) and IgG-9E10 (against Insig-1). The numbers to the side of immunoblots are referred to as panels in the text.
    Figure Legend Snippet: Reconstitution of lipid-regulated ERAD of mammalian Insig-1 in Drosophila S2 cells. S2 cells were set up in 6-well plates on day 0 at 1 × 10 6 cells per well in medium A supplemented with 10% HI-FCS. On day 1, cells were transfected in medium B using Maxfect™ as follows. A: 0.1 µg of pAc-Insig-1-myc or 0.1 µg of pAc-Insig-2-myc. B: 0.2 µg pAc-Insig-1-Myc in the absence or presence of 1.0 µg pAc-HA-ubiquitin. C: 0.1 µg of pAc-Insig-1-myc and 1, 3, 10, or 30 ng of pAc-Scap. D: 0.1 of µg pAc-Insig-1-myc. Total amount of DNA was adjusted to 0.1 μg (A, D), 1.2 μg (B), or 0.13 μg (C) using empty pAc5.1 vector. On day 2, each well received 1 ml of medium B supplemented with 20% HI-LPDS (A–C) or HI-DFCS (D). Cells were treated on day 3 with medium C supplemented with 10% HI-LPDS (A–C) or HI-DFCS (D) under the following conditions. A: In the absence or presence of 10 μM MG-132 (6 h) and 50 μM cycloheximide (2 h). B: In the presence of 10 µM MG-132 (2 h). C: In the absence and presence of 2.5 μM 25-HC and 10 mM mevalonate (4 h) together with 50 μM cycloheximide (2 h). D: In the absence and presence of 10 μM MG-132 (6 h) or 0.1, 0.3, or 1 mM BSA-oleate (4 h) together with 50 μM cycloheximide (2 h). A, C, D: Following incubations, cells were harvested and aliquots of whole cell lysates [30 μg protein/lane (A), 40 μg protein/lane (C), 50 μg protein/lane (D)] were subjected to 10% SDS-PAGE followed by immunoblot analysis with IgG-9E10 (against Insigs), IgG-9D5 (against hamster Scap), and anti-actin IgG. B: Following incubation, the cells were harvested for preparation of detergent lysates that were immunoprecipitated with 60 µl anti-Myc coupled agarose beads. Aliquots of the immunoprecipitates were then subjected to SDS-PAGE followed by immunoblot analysis with anti-HA (against ubiquitin) and IgG-9E10 (against Insig-1). The numbers to the side of immunoblots are referred to as panels in the text.

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

    15) Product Images from "Immunization with Staphylococcus aureus Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model"

    Article Title: Immunization with Staphylococcus aureus Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model

    Journal:

    doi: 10.1128/IAI.74.4.2145-2153.2006

    Effects of MAbs 3D6 and 6C5 on the attachment of S. aureus Newman (A) or biotinylated N2N3 of ClfB (B) to cytokeratin 10. The data are expressed as percentages of attached bacteria (A) or bound peptide (B) in the absence of MAbs. The values are means
    Figure Legend Snippet: Effects of MAbs 3D6 and 6C5 on the attachment of S. aureus Newman (A) or biotinylated N2N3 of ClfB (B) to cytokeratin 10. The data are expressed as percentages of attached bacteria (A) or bound peptide (B) in the absence of MAbs. The values are means

    Techniques Used:

    16) Product Images from "The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype"

    Article Title: The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039097

    rLCC111 substrates do not support cell attachment. Adhesion of HT0180 cells to plastic-coated LM111 or rLCC111 (ranging from 0.08 to 20 μg/ml), or BSA was measured by bioluminescence. (A). Data are plotted as the log of fold change in adhesion relative to BSA. The coating efficiency of LM111 and rLCC111 was monitored by ELISA using a polyclonal anti-laminin antibody (B). Soluble rLCC111 (5 to 20 mg/ml) inhibits adhesion of HT1080 cells (C) to plastic immobilized intact LM111 (10 mg/ml). Data shown are from a representative experiment out of three independent ones. (*, p
    Figure Legend Snippet: rLCC111 substrates do not support cell attachment. Adhesion of HT0180 cells to plastic-coated LM111 or rLCC111 (ranging from 0.08 to 20 μg/ml), or BSA was measured by bioluminescence. (A). Data are plotted as the log of fold change in adhesion relative to BSA. The coating efficiency of LM111 and rLCC111 was monitored by ELISA using a polyclonal anti-laminin antibody (B). Soluble rLCC111 (5 to 20 mg/ml) inhibits adhesion of HT1080 cells (C) to plastic immobilized intact LM111 (10 mg/ml). Data shown are from a representative experiment out of three independent ones. (*, p

    Techniques Used: Cell Attachment Assay, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Imaging of immunogold labeling in cells and tissues by helium ion microscopy"

    Article Title: Imaging of immunogold labeling in cells and tissues by helium ion microscopy

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3604

    Fluorescence identification of organelles imaged by SE-based HIM. To identify the organelles of COS7 cells, F-actin was labeled with Alexa Fluor ® 488-conjugated phalloidin (green), ER with anti-PDI antibody and further with Alexa Fluor ® 594-conjugated secondary antibody (red), and DNA with DAPI (blue). Labeling was checked in solution by fluorescence microscopy: (A) DAPI was used to identify the nucleus. (B) PDI was used to identify the ER. (C) F-actin was used to identify stress fibers. Cells were then dehydrated/dried and observed by SE-HIM. (D) SE-HIM, corresponding to C. (E) Magnification of the rectangle in D. The web-like network in the SE-HIM images corresponds to florescence signals from the ER (arrows), and the faint striated bundles to fluorescence signals from F-actin (arrowheads). DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; F-actin, filamentous-actin; HIM, helium ion microscopy; PDI, protein disulfide isomerase; SE, secondary electron.
    Figure Legend Snippet: Fluorescence identification of organelles imaged by SE-based HIM. To identify the organelles of COS7 cells, F-actin was labeled with Alexa Fluor ® 488-conjugated phalloidin (green), ER with anti-PDI antibody and further with Alexa Fluor ® 594-conjugated secondary antibody (red), and DNA with DAPI (blue). Labeling was checked in solution by fluorescence microscopy: (A) DAPI was used to identify the nucleus. (B) PDI was used to identify the ER. (C) F-actin was used to identify stress fibers. Cells were then dehydrated/dried and observed by SE-HIM. (D) SE-HIM, corresponding to C. (E) Magnification of the rectangle in D. The web-like network in the SE-HIM images corresponds to florescence signals from the ER (arrows), and the faint striated bundles to fluorescence signals from F-actin (arrowheads). DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; F-actin, filamentous-actin; HIM, helium ion microscopy; PDI, protein disulfide isomerase; SE, secondary electron.

    Techniques Used: Fluorescence, Labeling, Microscopy

    18) Product Images from "Internalization of novel non-viral vector TAT-streptavidin into human cells"

    Article Title: Internalization of novel non-viral vector TAT-streptavidin into human cells

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-1

    Quantification of internalized TAT-SA in living HeLa cells . (A) Quantitative analysis of the colocalization between both TAT-SA-A488 and TRITC-labeled transferrin and TRITC-labeled dextran (10 kD) at 15 min post transduction. (B) The relative fluorescence intensity of internalized TAT-SA-A488 was measured in cells treated with cytochalasin D (cytD), amiloride (ami) or nocodazole (noco) at 4 h post transduction. Control cells (C) were untreated. The fluorescence intensity data was collected from multiple series of cells by confocal microcopy and processed with the 3D LSM program.
    Figure Legend Snippet: Quantification of internalized TAT-SA in living HeLa cells . (A) Quantitative analysis of the colocalization between both TAT-SA-A488 and TRITC-labeled transferrin and TRITC-labeled dextran (10 kD) at 15 min post transduction. (B) The relative fluorescence intensity of internalized TAT-SA-A488 was measured in cells treated with cytochalasin D (cytD), amiloride (ami) or nocodazole (noco) at 4 h post transduction. Control cells (C) were untreated. The fluorescence intensity data was collected from multiple series of cells by confocal microcopy and processed with the 3D LSM program.

    Techniques Used: Labeling, Transduction, Fluorescence

    19) Product Images from "Antimalarial Quinolines and Artemisinin Inhibit Endocytosis in Plasmodium falciparum"

    Article Title: Antimalarial Quinolines and Artemisinin Inhibit Endocytosis in Plasmodium falciparum

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.7.2370-2378.2004

    RBC-free endocytosis assay. Parasites released from cultured pRBCs by saponin treatment were incubated for 5 h in a diluted RBC lysate containing FITC-dextran and no drug (A), 120 nM chloroquine (B), 1.5 μM chloroquine (C), mefloquine (D), artemisinin (E), ammonium chloride (F), or monensin (G). The parasites were washed, fixed, and viewed by fluorescence microscopy. Left panels, fluorescence images of individual parasites; right panels, the corresponding phase-contrast images. Arrows, transport vesicles; arrowheads, positions of the food vacuole. The percentages of parasites containing two or more transport vesicles are indicated in the bottom left corner of the phase-contrast panels.
    Figure Legend Snippet: RBC-free endocytosis assay. Parasites released from cultured pRBCs by saponin treatment were incubated for 5 h in a diluted RBC lysate containing FITC-dextran and no drug (A), 120 nM chloroquine (B), 1.5 μM chloroquine (C), mefloquine (D), artemisinin (E), ammonium chloride (F), or monensin (G). The parasites were washed, fixed, and viewed by fluorescence microscopy. Left panels, fluorescence images of individual parasites; right panels, the corresponding phase-contrast images. Arrows, transport vesicles; arrowheads, positions of the food vacuole. The percentages of parasites containing two or more transport vesicles are indicated in the bottom left corner of the phase-contrast panels.

    Techniques Used: Endocytosis Assay, Cell Culture, Incubation, Fluorescence, Microscopy

    Quantitative endocytosis assays. (A and B) RBCs were preloaded with biotinylated dextran; infected with parasites; and treated with chloroquine (CQ), mefloquine (MEF), or artemisinin (ART) or were left untreated as controls (CON) for 12 h (A) or 5 h (B). Subsequently, the parasites were released from their host RBCs by saponin treatment, and their biotin-dextran contents were determined by incubating parasite lysates with streptavidin-coated microtiter plates, followed by incubation with streptavidin-horseradish peroxidase and colorimetric peroxidase substrate. (C) Parasites released from RBCs by saponin lysis were incubated with FITC-dextran in the presence of the indicated antimalarials, fixed, and viewed by fluorescence microscopy. Images of individual parasites were captured with a cooled charge-coupled device camera, and the mean fluorescence intensity in the food vacuoles was determined with Adobe Photoshop software. *, P
    Figure Legend Snippet: Quantitative endocytosis assays. (A and B) RBCs were preloaded with biotinylated dextran; infected with parasites; and treated with chloroquine (CQ), mefloquine (MEF), or artemisinin (ART) or were left untreated as controls (CON) for 12 h (A) or 5 h (B). Subsequently, the parasites were released from their host RBCs by saponin treatment, and their biotin-dextran contents were determined by incubating parasite lysates with streptavidin-coated microtiter plates, followed by incubation with streptavidin-horseradish peroxidase and colorimetric peroxidase substrate. (C) Parasites released from RBCs by saponin lysis were incubated with FITC-dextran in the presence of the indicated antimalarials, fixed, and viewed by fluorescence microscopy. Images of individual parasites were captured with a cooled charge-coupled device camera, and the mean fluorescence intensity in the food vacuoles was determined with Adobe Photoshop software. *, P

    Techniques Used: Infection, Incubation, Lysis, Fluorescence, Microscopy, Software

    Hemoglobin accumulation in parasites. Parasite cultures were left untreated (control; C) or were incubated with chloroquine (CQ), mefloquine (MQ), or artemisinin (ART) for 12 h (A) or 5 h (B). Parasites were released from RBCs by saponin treatment and were analyzed for their hemoglobin contents by Western blotting with an antihemoglobin antiserum. The net intensity of the individual hemoglobin bands was determined with Kodak 1D image analysis software. The intensity values indicated are normalized values ± SD obtained from four different experiments in each case.
    Figure Legend Snippet: Hemoglobin accumulation in parasites. Parasite cultures were left untreated (control; C) or were incubated with chloroquine (CQ), mefloquine (MQ), or artemisinin (ART) for 12 h (A) or 5 h (B). Parasites were released from RBCs by saponin treatment and were analyzed for their hemoglobin contents by Western blotting with an antihemoglobin antiserum. The net intensity of the individual hemoglobin bands was determined with Kodak 1D image analysis software. The intensity values indicated are normalized values ± SD obtained from four different experiments in each case.

    Techniques Used: Incubation, Western Blot, Software

    Parasite endocytosis of FITC-dextran from preloaded RBCs. RBCs preloaded with FITC-dextran and infected with parasites were left untreated (A) or were treated with chloroquine (B), artemisinin (C), or mefloquine (D) for 12 h. The pRBCs were subsequently immobilized on glass coverslips, rinsed in saponin, fixed, and viewed by fluorescence microscopy. Left panels, fluorescence images; right panels, corresponding phase-contrast images. Arrows denote the locations of the food vacuole, as indicated by the presence of the prominent hemozoin crystal.
    Figure Legend Snippet: Parasite endocytosis of FITC-dextran from preloaded RBCs. RBCs preloaded with FITC-dextran and infected with parasites were left untreated (A) or were treated with chloroquine (B), artemisinin (C), or mefloquine (D) for 12 h. The pRBCs were subsequently immobilized on glass coverslips, rinsed in saponin, fixed, and viewed by fluorescence microscopy. Left panels, fluorescence images; right panels, corresponding phase-contrast images. Arrows denote the locations of the food vacuole, as indicated by the presence of the prominent hemozoin crystal.

    Techniques Used: Infection, Fluorescence, Microscopy

    20) Product Images from "A Functional Heat Shock Protein 90 Chaperone Is Essential for Efficient Flock House Virus RNA Polymerase Synthesis in Drosophila Cells ▿"

    Article Title: A Functional Heat Shock Protein 90 Chaperone Is Essential for Efficient Flock House Virus RNA Polymerase Synthesis in Drosophila Cells ▿

    Journal:

    doi: 10.1128/JVI.00189-07

    Hsp90 inhibition does not alter FHV protein A membrane association in Drosophila S2 cells. (A) Schematic of pulse-chase fractionation protocol. (B) Cells stably transfected with pS2FA or pS2LacZ were induced with Cu 2+ ; pulse labeled with 500 μCi
    Figure Legend Snippet: Hsp90 inhibition does not alter FHV protein A membrane association in Drosophila S2 cells. (A) Schematic of pulse-chase fractionation protocol. (B) Cells stably transfected with pS2FA or pS2LacZ were induced with Cu 2+ ; pulse labeled with 500 μCi

    Techniques Used: Inhibition, Pulse Chase, Fractionation, Stable Transfection, Transfection, Labeling

    Newly synthesized FHV protein A rapidly associates with cellular membranes in Drosophila S2 cells. (A) Cells were incubated with PBS and 0.01% saponin on ice for 10 min and centrifuged at 10,000 × g for 5 min to recover the supernatant
    Figure Legend Snippet: Newly synthesized FHV protein A rapidly associates with cellular membranes in Drosophila S2 cells. (A) Cells were incubated with PBS and 0.01% saponin on ice for 10 min and centrifuged at 10,000 × g for 5 min to recover the supernatant

    Techniques Used: Synthesized, Incubation

    Hsp90 inhibition reduces FHV protein A mRNA association with polysomes. (A) Drosophila S2 cells stably transfected with pS2FA were induced with Cu 2+ in the presence of dimethyl sulfoxide (DMSO) or geldanamycin (GA), cell lysates were separated
    Figure Legend Snippet: Hsp90 inhibition reduces FHV protein A mRNA association with polysomes. (A) Drosophila S2 cells stably transfected with pS2FA were induced with Cu 2+ in the presence of dimethyl sulfoxide (DMSO) or geldanamycin (GA), cell lysates were separated

    Techniques Used: Inhibition, Stable Transfection, Transfection

    Hsp90 inhibition suppresses FHV protein A synthesis. (A) Drosophila S2 cells stably transfected with pS2FA (lanes 1 to 4), pS2LacZ (lanes 5 to 7), or pS2FB (lanes 8 and 9) were induced with Cu 2+ and labeled with 100 μCi per ml [ 35 S]Met-Cys
    Figure Legend Snippet: Hsp90 inhibition suppresses FHV protein A synthesis. (A) Drosophila S2 cells stably transfected with pS2FA (lanes 1 to 4), pS2LacZ (lanes 5 to 7), or pS2FB (lanes 8 and 9) were induced with Cu 2+ and labeled with 100 μCi per ml [ 35 S]Met-Cys

    Techniques Used: Inhibition, Stable Transfection, Transfection, Labeling

    mRNA 5′ UTR does not affect geldanamycin-mediated suppression of FHV protein A synthesis. (A) Schematics of pS2FA and derivatives with modified 5′ UTRs. All plasmids contain an MT promoter and a simian virus 40 polyadenylation signal (
    Figure Legend Snippet: mRNA 5′ UTR does not affect geldanamycin-mediated suppression of FHV protein A synthesis. (A) Schematics of pS2FA and derivatives with modified 5′ UTRs. All plasmids contain an MT promoter and a simian virus 40 polyadenylation signal (

    Techniques Used: Modification

    Hsp90 suppression does not accelerate FHV protein A degradation. (A) Basal FHV protein A stability in transfected Drosophila S2 cells. Cells stably transfected with pS2FA were induced with Cu 2+ and labeled with 125 μCi per ml [ 35 S]Met-Cys
    Figure Legend Snippet: Hsp90 suppression does not accelerate FHV protein A degradation. (A) Basal FHV protein A stability in transfected Drosophila S2 cells. Cells stably transfected with pS2FA were induced with Cu 2+ and labeled with 125 μCi per ml [ 35 S]Met-Cys

    Techniques Used: Transfection, Stable Transfection, Labeling

    Hsp90 inhibition suppresses the synthesis of retargeted FHV protein A. (A) Schematics of pS2FA, which encodes wild-type mitochondrial targeted protein A, and pS2FA-P450, which encodes a chimeric protein A retargeted to the endoplasmic reticulum. The amino-terminal
    Figure Legend Snippet: Hsp90 inhibition suppresses the synthesis of retargeted FHV protein A. (A) Schematics of pS2FA, which encodes wild-type mitochondrial targeted protein A, and pS2FA-P450, which encodes a chimeric protein A retargeted to the endoplasmic reticulum. The amino-terminal

    Techniques Used: Inhibition

    21) Product Images from "Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation"

    Article Title: Suppression of experimental arthritis through AMP-activated protein kinase activation and autophagy modulation

    Journal: Journal of rheumatic diseases and treatment

    doi:

    Metformin enhances autophagic flux (A) Paw lysates from normal and day 9 KRN serum-induced arthritic mice were probed for LC3-I, the lipidated form LC3-II, and p62. Actin served as control for protein loading. (B) Paw sections from untreated arthritic mice or arthritic mice treated with saline or metformin were stained for LC3 (green) and the lysosomal marker LAMP-1 (red). Note the punctate LC3 staining in untreated or saline control arthritic paws. Colocalization of LC3 and LAMP-1 (arrow) indicates autolysosome. Scale bar = 10 μm. (C) Treatment with metformin led to degradation of the lipidated LC3-II form and p62, indicating enhanced autophagic flux. (D) Day 9 paws were stained for phospho (p)-ULK1 Ser555 (red). DAPI (blue) stained nuclei. Scale bar = 25 μm. (E) Intracellular level of p-ULK1was analyzed using ImageJ program as detailed in the Materials and Methods section and presented as integrated optical density (IntDen). Values represent mean ± SEM, n = 4–5 mice per treatment group. * P
    Figure Legend Snippet: Metformin enhances autophagic flux (A) Paw lysates from normal and day 9 KRN serum-induced arthritic mice were probed for LC3-I, the lipidated form LC3-II, and p62. Actin served as control for protein loading. (B) Paw sections from untreated arthritic mice or arthritic mice treated with saline or metformin were stained for LC3 (green) and the lysosomal marker LAMP-1 (red). Note the punctate LC3 staining in untreated or saline control arthritic paws. Colocalization of LC3 and LAMP-1 (arrow) indicates autolysosome. Scale bar = 10 μm. (C) Treatment with metformin led to degradation of the lipidated LC3-II form and p62, indicating enhanced autophagic flux. (D) Day 9 paws were stained for phospho (p)-ULK1 Ser555 (red). DAPI (blue) stained nuclei. Scale bar = 25 μm. (E) Intracellular level of p-ULK1was analyzed using ImageJ program as detailed in the Materials and Methods section and presented as integrated optical density (IntDen). Values represent mean ± SEM, n = 4–5 mice per treatment group. * P

    Techniques Used: Mouse Assay, Staining, Marker

    Metformin promotes selective degradation of NF-κB protein Iκ kinase (IKK) through autophagy (A) Day 9 paw lysates from saline controls and metformin-treated animals were probed for NF-κB proteins (IKKα, IκB-β, p65, and phospho-p65). Actin served as protein loading control. (B) Day 9 paw sections were probed for ubiquitin (green) and p62 (red). Colocalization (arrowheads, yellow) suggests ubiquinated-p62 aggregates. (C) Equivalent amounts of protein were immunoprecipitated with anti-ubiquitin antibody and probed with anti-IKKα antibody. High molecular weight protein complexes likely represent poly-ubiquinated IKKα. (D) Paw sections were also probed for IKKα and LAMP-1. Accumulation of IKKα was evident in the saline controls while IKKα level was significantly lower in metformin-treated animals. Colocalization of IKKα and LAMP-1 (arrows) in metformin treatment suggests IKKα targeted to the lysosomes.
    Figure Legend Snippet: Metformin promotes selective degradation of NF-κB protein Iκ kinase (IKK) through autophagy (A) Day 9 paw lysates from saline controls and metformin-treated animals were probed for NF-κB proteins (IKKα, IκB-β, p65, and phospho-p65). Actin served as protein loading control. (B) Day 9 paw sections were probed for ubiquitin (green) and p62 (red). Colocalization (arrowheads, yellow) suggests ubiquinated-p62 aggregates. (C) Equivalent amounts of protein were immunoprecipitated with anti-ubiquitin antibody and probed with anti-IKKα antibody. High molecular weight protein complexes likely represent poly-ubiquinated IKKα. (D) Paw sections were also probed for IKKα and LAMP-1. Accumulation of IKKα was evident in the saline controls while IKKα level was significantly lower in metformin-treated animals. Colocalization of IKKα and LAMP-1 (arrows) in metformin treatment suggests IKKα targeted to the lysosomes.

    Techniques Used: Immunoprecipitation, Molecular Weight

    22) Product Images from "?-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts V⃞"

    Article Title: ?-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts V⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-11-0729

    The adhesion capacity of fibroblasts in long-term culture correlates with their level of α-SMA expression and the degree of FA supermaturation. REF-52 were grown for 5 d in culture medium containing 20% FCS (A), 10% FCS (B and D), and 2% FCS (C). (A–C) Cells were immunostained for α-SMA (blue), vinculin (green), and β-cytoplasmic actin (red) and observed by confocal microscopy; shift from red to purple indicates increase in α-SMA expression. (D) Cells were stained for α5β1 integrin (green), tensin (blue), and β3 integrin (red); tensin colocalizes with β3 integrin in supermature FAs (pink) and with α5β1 integrin in fibrillar adhesions (turquoise). Bar, 25 μm. (E) REF-52 were plated for 5 d in different serum conditions, and protein expression was assessed by Western blotting. The expression levels of α-SMA, paxillin, vinculin, and β3 integrin increase with decreasing serum concentration. To test their adhesion capacity, REF-52 with different levels of α-SMA expression were trypsinized and plated for 2 d. After treatment with FPs and 0.02% EDTA for 60 min, weakly adherent cells were removed by centrifugation. Cell adhesion increases with increasing α-SMA expression. SMA-FP reduces the adhesion of α-SMA-expressing fibroblasts to the level of α-SMA–negative fibroblasts; SKA-FP has no effect.
    Figure Legend Snippet: The adhesion capacity of fibroblasts in long-term culture correlates with their level of α-SMA expression and the degree of FA supermaturation. REF-52 were grown for 5 d in culture medium containing 20% FCS (A), 10% FCS (B and D), and 2% FCS (C). (A–C) Cells were immunostained for α-SMA (blue), vinculin (green), and β-cytoplasmic actin (red) and observed by confocal microscopy; shift from red to purple indicates increase in α-SMA expression. (D) Cells were stained for α5β1 integrin (green), tensin (blue), and β3 integrin (red); tensin colocalizes with β3 integrin in supermature FAs (pink) and with α5β1 integrin in fibrillar adhesions (turquoise). Bar, 25 μm. (E) REF-52 were plated for 5 d in different serum conditions, and protein expression was assessed by Western blotting. The expression levels of α-SMA, paxillin, vinculin, and β3 integrin increase with decreasing serum concentration. To test their adhesion capacity, REF-52 with different levels of α-SMA expression were trypsinized and plated for 2 d. After treatment with FPs and 0.02% EDTA for 60 min, weakly adherent cells were removed by centrifugation. Cell adhesion increases with increasing α-SMA expression. SMA-FP reduces the adhesion of α-SMA-expressing fibroblasts to the level of α-SMA–negative fibroblasts; SKA-FP has no effect.

    Techniques Used: Expressing, Confocal Microscopy, Staining, Western Blot, Concentration Assay, Centrifugation

    Filament bundles of early spreading myofibroblasts contain only β-cytoplasmic actin. REF-52 fibroblasts were trypsinized and plated for 2 h (A–C) and 3 h (D–F) and then fixed and triple-immunostained against β-cytoplasmic actin (A and D), α-SMA (B and E), and vinculin (C and F). Insets show at high magnification diffuse organization of α-SMA in the presence of β-cytoplasmic actin filament bundles. Incorporation of α-SMA into stress fibers starts 3 h after plating, correlating with FA maturation and cell polarization. Bar, 50 μm.
    Figure Legend Snippet: Filament bundles of early spreading myofibroblasts contain only β-cytoplasmic actin. REF-52 fibroblasts were trypsinized and plated for 2 h (A–C) and 3 h (D–F) and then fixed and triple-immunostained against β-cytoplasmic actin (A and D), α-SMA (B and E), and vinculin (C and F). Insets show at high magnification diffuse organization of α-SMA in the presence of β-cytoplasmic actin filament bundles. Incorporation of α-SMA into stress fibers starts 3 h after plating, correlating with FA maturation and cell polarization. Bar, 50 μm.

    Techniques Used:

    23) Product Images from "Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism"

    Article Title: Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.16-17-05425.1996

    Transient transfection of the β-galactosidase gene into primary cortical neurons using a modified calcium phosphate procedure. A , X-Gal staining ( blue ) of cortical neurons (P0) transfected at 3 DIV with an expression vector encoding β-galactosidase. B , A representative immunofluorescence photomicrograph of a neuron transfected with β-galactosidase. Transfected cortical neurons cultured from E17/18 rats were detected by immunostaining with a monoclonal antibody to β-galactosidase, visualized by fluorescein-conjugated goat antibody to mouse IgG ( green ). The cells were coimmunostained for the neuronal marker protein MAP-2, which was visualized using a Texas Red-conjugated goat antibody to rabbit IgG ( red ). The transfected neuron appears yellow because of the colocalization of green staining from anti-β-galactosidase and red staining from anti-MAP-2. C , A representative immunofluorescence photomicrograph of a healthy neuron expressing β-galactosidase. A transfected E17/18 rat cortical neuron was detected as in B . The nucleus was visualized using the DNA dye Hoechst 33258 ( blue ). The evenly stained, round morphology is typical of a healthy (nonapoptotic) nucleus.
    Figure Legend Snippet: Transient transfection of the β-galactosidase gene into primary cortical neurons using a modified calcium phosphate procedure. A , X-Gal staining ( blue ) of cortical neurons (P0) transfected at 3 DIV with an expression vector encoding β-galactosidase. B , A representative immunofluorescence photomicrograph of a neuron transfected with β-galactosidase. Transfected cortical neurons cultured from E17/18 rats were detected by immunostaining with a monoclonal antibody to β-galactosidase, visualized by fluorescein-conjugated goat antibody to mouse IgG ( green ). The cells were coimmunostained for the neuronal marker protein MAP-2, which was visualized using a Texas Red-conjugated goat antibody to rabbit IgG ( red ). The transfected neuron appears yellow because of the colocalization of green staining from anti-β-galactosidase and red staining from anti-MAP-2. C , A representative immunofluorescence photomicrograph of a healthy neuron expressing β-galactosidase. A transfected E17/18 rat cortical neuron was detected as in B . The nucleus was visualized using the DNA dye Hoechst 33258 ( blue ). The evenly stained, round morphology is typical of a healthy (nonapoptotic) nucleus.

    Techniques Used: Transfection, Modification, Staining, Expressing, Plasmid Preparation, Immunofluorescence, Cell Culture, Immunostaining, Marker

    24) Product Images from "L1 Antibodies Block Lymph Node Fibroblastic Reticular Matrix Remodeling In Vivo "

    Article Title: L1 Antibodies Block Lymph Node Fibroblastic Reticular Matrix Remodeling In Vivo

    Journal: The Journal of Experimental Medicine

    doi:

    Histological and immunological examination of FRS architecture in immune LNs. Controls A , C , and E were harvested from mice injected with the NRIg. B , D , and F are LNs from L1 mAb–injected animals. ( A and B ) Stacked confocal images of LN sections cut 48 μm thick and immunostained for vimentin. The RF processes stain brightest for vimentin and appear white against the dark red background. ( C and D ) Photomicrographs of 6-μm-thick frozen LN sections stained for laminin by immunohistochemistry. V , Venules. C , Capillaries. L , Laminin. ( E and F ) Photomicrographs of toluidine blue–stained, 1-μm-thick, Araldite-embedded LN sections. E demonstrates normal compartmentalization of an NRIg-treated control LN. N , Lymph nodule. V , Venule. S , Sinus. Scs , Subcapsular sinus. F illustrates the loss of nodular architecture and collapse of cortical sinuses in LNs from L1 mAb–treated animals. In F , the structure designated with a V is a venule, not a tangential cut of a sinusoid, as evidenced by the erythrocytes within its lumen (not easily visible at this low power). Identical results were obtained using both NRIg and anti-class I as control antibodies in experiments used for plastic embedding. Bars, 10 μm in A–D , 20 μm in E and F .
    Figure Legend Snippet: Histological and immunological examination of FRS architecture in immune LNs. Controls A , C , and E were harvested from mice injected with the NRIg. B , D , and F are LNs from L1 mAb–injected animals. ( A and B ) Stacked confocal images of LN sections cut 48 μm thick and immunostained for vimentin. The RF processes stain brightest for vimentin and appear white against the dark red background. ( C and D ) Photomicrographs of 6-μm-thick frozen LN sections stained for laminin by immunohistochemistry. V , Venules. C , Capillaries. L , Laminin. ( E and F ) Photomicrographs of toluidine blue–stained, 1-μm-thick, Araldite-embedded LN sections. E demonstrates normal compartmentalization of an NRIg-treated control LN. N , Lymph nodule. V , Venule. S , Sinus. Scs , Subcapsular sinus. F illustrates the loss of nodular architecture and collapse of cortical sinuses in LNs from L1 mAb–treated animals. In F , the structure designated with a V is a venule, not a tangential cut of a sinusoid, as evidenced by the erythrocytes within its lumen (not easily visible at this low power). Identical results were obtained using both NRIg and anti-class I as control antibodies in experiments used for plastic embedding. Bars, 10 μm in A–D , 20 μm in E and F .

    Techniques Used: Mouse Assay, Injection, Staining, Immunohistochemistry

    25) Product Images from "Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound"

    Article Title: Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2013.05.008

    MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p
    Figure Legend Snippet: MRIgFUS treatment causes a reduction in Aβ plaque pathology. Plaque load was assessed 4 days following MRIgFUS treatment using immunohistochemistry and stereology. A , Plaques were quantified in MRIgFUS-treated (right) and an equivalent untreated (left) region of the cortex. Significant reductions were observed in mean plaque size ( B ) and total Aβ surface area ( C ) in the MRIgFUS-treated compared to untreated cortex. D , Plaque counts within the MRIgFUS-targeted region demonstrated a trend of reduction compared to the untreated region. Within the MRIgFUS-treated cortex, endogenous IgG ( F ) and IgM ( G ), were found to be co-localized with plaques ( E ). At the site of plaques in the untreated cortex ( H ), a small amount of IgG ( I ) but no IgM ( J ) was found. White arrows indicate the location of Aβ plaques (E–J). Scale bar: A=0.5 mm, E–J = 50 μm. Data is presented as paired values between left, untreated and right, MRIgFUS-treated, *p

    Techniques Used: Immunohistochemistry

    MRIgFUS facilitates the entry of IgG and IgM into the brain. Using immunohistochemistry, high levels of IgG were detected in MRIgFUS-treated compared to untreated cortex of TgCRND8 ( A ) and non-Tg ( B ) mice. Similarly, IgM observed in MRIgFUS-treated cortical regions was greater than that detected in contralateral cortex of TgCRND8 ( C ) and non-Tg mice ( D ). Quantitative western blot analysis revealed that compared to untreated cortex, the cortex treated with MRIgFUS had greater levels of IgG in TgCRND8 ( E ) and non-Tg ( F ) mice; and of IgM in TgCRND8 ( G ) and non-Tg ( H ) mice. Scale bar: A–D= 500 μm. Data are presented as the mean ± SEM, *p
    Figure Legend Snippet: MRIgFUS facilitates the entry of IgG and IgM into the brain. Using immunohistochemistry, high levels of IgG were detected in MRIgFUS-treated compared to untreated cortex of TgCRND8 ( A ) and non-Tg ( B ) mice. Similarly, IgM observed in MRIgFUS-treated cortical regions was greater than that detected in contralateral cortex of TgCRND8 ( C ) and non-Tg mice ( D ). Quantitative western blot analysis revealed that compared to untreated cortex, the cortex treated with MRIgFUS had greater levels of IgG in TgCRND8 ( E ) and non-Tg ( F ) mice; and of IgM in TgCRND8 ( G ) and non-Tg ( H ) mice. Scale bar: A–D= 500 μm. Data are presented as the mean ± SEM, *p

    Techniques Used: Immunohistochemistry, Mouse Assay, Western Blot

    26) Product Images from "Imaging of immunogold labeling in cells and tissues by helium ion microscopy"

    Article Title: Imaging of immunogold labeling in cells and tissues by helium ion microscopy

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3604

    Fluorescence identification of organelles imaged by SE-based HIM. To identify the organelles of COS7 cells, F-actin was labeled with Alexa Fluor ® 488-conjugated phalloidin (green), ER with anti-PDI antibody and further with Alexa Fluor ® 594-conjugated secondary antibody (red), and DNA with DAPI (blue). Labeling was checked in solution by fluorescence microscopy: (A) DAPI was used to identify the nucleus. (B) PDI was used to identify the ER. (C) F-actin was used to identify stress fibers. Cells were then dehydrated/dried and observed by SE-HIM. (D) SE-HIM, corresponding to C. (E) Magnification of the rectangle in D. The web-like network in the SE-HIM images corresponds to florescence signals from the ER (arrows), and the faint striated bundles to fluorescence signals from F-actin (arrowheads). DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; F-actin, filamentous-actin; HIM, helium ion microscopy; PDI, protein disulfide isomerase; SE, secondary electron.
    Figure Legend Snippet: Fluorescence identification of organelles imaged by SE-based HIM. To identify the organelles of COS7 cells, F-actin was labeled with Alexa Fluor ® 488-conjugated phalloidin (green), ER with anti-PDI antibody and further with Alexa Fluor ® 594-conjugated secondary antibody (red), and DNA with DAPI (blue). Labeling was checked in solution by fluorescence microscopy: (A) DAPI was used to identify the nucleus. (B) PDI was used to identify the ER. (C) F-actin was used to identify stress fibers. Cells were then dehydrated/dried and observed by SE-HIM. (D) SE-HIM, corresponding to C. (E) Magnification of the rectangle in D. The web-like network in the SE-HIM images corresponds to florescence signals from the ER (arrows), and the faint striated bundles to fluorescence signals from F-actin (arrowheads). DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; F-actin, filamentous-actin; HIM, helium ion microscopy; PDI, protein disulfide isomerase; SE, secondary electron.

    Techniques Used: Fluorescence, Labeling, Microscopy

    27) Product Images from "Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38? Activation"

    Article Title: Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38? Activation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.16.6957-6966.2004

    Anergic CD4 + T cells express TAB1. (A) Naive and anergic CD4 + T cells were purified by using BD IMag-anti-mouse CD4 MAb, cultured with or without SB203580 (10 μM) for 2 h before stimulation, and plated in wells coated with anti-TCR MAb (10 μg/ml) for 0, 15, or 30 min. Cells were harvested and lysed, and the levels of phospho-p38 and total p38α were determined by immunoblotting with specific antibodies. Representative results from three independent experiments are shown. (B) Lysates from naive and anergic CD4 + T cells (4 × 10 6 /lane) were separated by SDS-12.5% PAGE and examined for the presence of TAB1 by immunoblotting with specific antibodies. After stripping, the membrane was immunoblotted with antiactin antibody. rTAB1 1-312 , N-terminal 312-amino-acid fragment of recombinant TAB1. (C) Expression of TAB1 during the induction of T-cell anergy. CD4 + T cells were purified from individual mice on the indicated days after Mls-1 a inoculation. Lysates from two samples per group were separated by SDS-12.5% PAGE and examined for the expression of TAB1 by immunoblotting. Representative results from two independent experiments are shown. (D) CD4 + T cells from OT-II mice were cultured for 7 days in Th1 and Th2 mixtures. Anergic CD4 + T cells were prepared from Vβ8.1-tg mice 14 days after Mls-1 a inoculation (anergic), RNA was extracted from each cell type, and RT-PCR analysis of T-bet, GATA3, TAB1, and G3PDH was performed as described in Materials and Methods. (E) CD4 + T cells were prepared from Vβ8.1-tg mice (naive). Anergic, Th1, and Th2 cells were prepared as described for panel D. CD4 + T cells from Vβ8.1-tg mice were stimulated with anti-TCR MAb (0.5 μg/ml) and soluble anti-CD28 MAb (1 μg/ml) for 24 h in vitro (activated). Total cell lysates were separated by SDS-12.5% PAGE, blotted, and probed with anti-TAB1 antibody. A recombinant TAB1 fragment (rTAB1 1-312 ) was used as a positive control.
    Figure Legend Snippet: Anergic CD4 + T cells express TAB1. (A) Naive and anergic CD4 + T cells were purified by using BD IMag-anti-mouse CD4 MAb, cultured with or without SB203580 (10 μM) for 2 h before stimulation, and plated in wells coated with anti-TCR MAb (10 μg/ml) for 0, 15, or 30 min. Cells were harvested and lysed, and the levels of phospho-p38 and total p38α were determined by immunoblotting with specific antibodies. Representative results from three independent experiments are shown. (B) Lysates from naive and anergic CD4 + T cells (4 × 10 6 /lane) were separated by SDS-12.5% PAGE and examined for the presence of TAB1 by immunoblotting with specific antibodies. After stripping, the membrane was immunoblotted with antiactin antibody. rTAB1 1-312 , N-terminal 312-amino-acid fragment of recombinant TAB1. (C) Expression of TAB1 during the induction of T-cell anergy. CD4 + T cells were purified from individual mice on the indicated days after Mls-1 a inoculation. Lysates from two samples per group were separated by SDS-12.5% PAGE and examined for the expression of TAB1 by immunoblotting. Representative results from two independent experiments are shown. (D) CD4 + T cells from OT-II mice were cultured for 7 days in Th1 and Th2 mixtures. Anergic CD4 + T cells were prepared from Vβ8.1-tg mice 14 days after Mls-1 a inoculation (anergic), RNA was extracted from each cell type, and RT-PCR analysis of T-bet, GATA3, TAB1, and G3PDH was performed as described in Materials and Methods. (E) CD4 + T cells were prepared from Vβ8.1-tg mice (naive). Anergic, Th1, and Th2 cells were prepared as described for panel D. CD4 + T cells from Vβ8.1-tg mice were stimulated with anti-TCR MAb (0.5 μg/ml) and soluble anti-CD28 MAb (1 μg/ml) for 24 h in vitro (activated). Total cell lysates were separated by SDS-12.5% PAGE, blotted, and probed with anti-TAB1 antibody. A recombinant TAB1 fragment (rTAB1 1-312 ) was used as a positive control.

    Techniques Used: Purification, Cell Culture, Polyacrylamide Gel Electrophoresis, Stripping Membranes, Recombinant, Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Vitro, Positive Control

    28) Product Images from "IgG subclass response to Helicobacter pylori and CagA antigens in children"

    Article Title: IgG subclass response to Helicobacter pylori and CagA antigens in children

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2003.02304.x

    CagA IgG Units in H. pylori infected children with active ( n = 67) versus inactive ( n = 8) gastritis (Mann–Whitney U -test, P
    Figure Legend Snippet: CagA IgG Units in H. pylori infected children with active ( n = 67) versus inactive ( n = 8) gastritis (Mann–Whitney U -test, P

    Techniques Used: Infection, MANN-WHITNEY

    29) Product Images from "Misguided Axonal Projections, Neural Cell Adhesion Molecule 180 mRNA Upregulation, and Altered Behavior in Mice Deficient for the Close Homolog of L1"

    Article Title: Misguided Axonal Projections, Neural Cell Adhesion Molecule 180 mRNA Upregulation, and Altered Behavior in Mice Deficient for the Close Homolog of L1

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.22.7967-7981.2002

    Mossy fiber organization in adult CHL1-deficient mice. Sagittal sections stained for the presence of calbindin (green in panels A and B; mossy fiber axons) and synaptophysin (red in panels A to D; mossy fiber synaptic boutons). Conventional fluorescence microscopy (A and B) showing that in wild-type mice (A) the mossy fibers are orientated strictly parallel to the pyramidal cell layer; in contrast, in CHL1-deficient mice (B) many small bundles or individual thin mossy fibers travel through the CA3 region. Higher-magnification confocal microscopy showing the distribution of the mossy fiber terminals in the CA3 subfield of the hippocampus. (C) In wild-type mice the mossy fiber synaptic boutons are organized in a laminated pattern, only few synapses are detectable within the pyramidal cell layer (Py); the stratum lucidum (SL) can be clearly distinguished from the stratum pyramidale. (D) In CHL1-deficient mice, mossy fiber terminals are also detected throughout the CA3 pyramidal cell body layer surrounding the pyramidal cell soma (arrows). Scale bars, 200 μm (A and B) and 50 μm (C and D).
    Figure Legend Snippet: Mossy fiber organization in adult CHL1-deficient mice. Sagittal sections stained for the presence of calbindin (green in panels A and B; mossy fiber axons) and synaptophysin (red in panels A to D; mossy fiber synaptic boutons). Conventional fluorescence microscopy (A and B) showing that in wild-type mice (A) the mossy fibers are orientated strictly parallel to the pyramidal cell layer; in contrast, in CHL1-deficient mice (B) many small bundles or individual thin mossy fibers travel through the CA3 region. Higher-magnification confocal microscopy showing the distribution of the mossy fiber terminals in the CA3 subfield of the hippocampus. (C) In wild-type mice the mossy fiber synaptic boutons are organized in a laminated pattern, only few synapses are detectable within the pyramidal cell layer (Py); the stratum lucidum (SL) can be clearly distinguished from the stratum pyramidale. (D) In CHL1-deficient mice, mossy fiber terminals are also detected throughout the CA3 pyramidal cell body layer surrounding the pyramidal cell soma (arrows). Scale bars, 200 μm (A and B) and 50 μm (C and D).

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

    30) Product Images from "Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways"

    Article Title: Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092710

    Chicken LILRA2 and LILRA6 regulate the JAK-STAT signaling pathway. ( A ) Western blot of phosphorylated and un-phosphorylated STAT1, STAT3, JAK2 and TYK2 in LILRA2 and LILRA6 transfected HD11 cells; ( B ) qRT-PCR expression of STAT1, STAT3, JAK2 and TYK2 in LILRA2 and LILRA6 transfected HD11 cells; ( C ) FACS analysis of phosphorylated STAT1, STAT3 and JAK2 in LILRA2 and LILRA6 transfected HD11 cells. Data are presented as the mean ± SEM of three independent experiments: * p
    Figure Legend Snippet: Chicken LILRA2 and LILRA6 regulate the JAK-STAT signaling pathway. ( A ) Western blot of phosphorylated and un-phosphorylated STAT1, STAT3, JAK2 and TYK2 in LILRA2 and LILRA6 transfected HD11 cells; ( B ) qRT-PCR expression of STAT1, STAT3, JAK2 and TYK2 in LILRA2 and LILRA6 transfected HD11 cells; ( C ) FACS analysis of phosphorylated STAT1, STAT3 and JAK2 in LILRA2 and LILRA6 transfected HD11 cells. Data are presented as the mean ± SEM of three independent experiments: * p

    Techniques Used: Western Blot, Transfection, Quantitative RT-PCR, Expressing, FACS

    31) Product Images from "Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK1 cells"

    Article Title: Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK1 cells

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00228.2018

    Characterization of synapse-associated protein-97 (SAP97) and aquaporin-2 (AQP2) interactions in LLC-PK 1 cells and in mouse kidneys. A and B : coimmunoprecipitations (co-IPs) between c-Myc-AQP2 and SAP97. A : anti-c-Myc resin ( lane 2 ) but not preimmune IgG ( lane 3 ) coimmunoprecipitated SAP97 from c-Myc-AQP2-expressing cells. B : anti-SAP97 IgG resin coimmunoprecipitated AQP2 from c-Myc-AQP2-expressing cells. C : co-IPs between AQP2∆PDZ and SAP97. Inputs ( lane 1 ) represent ~4% of total cell lysate from each condition. D : LLC-PK 1 cells expressing c-Myc-WT AQP2 (pseudo red) and SAP97-green fluorescent protein (GFP) (pseudo green) were fixed, permeabilized, stained with CF555 anti-c-Myc IgG, and visualized by dual confocal microscopy. E : medullary slices from C57 black mouse kidneys were fixed, incubated with monoclonal anti-SAP97 and rabbit anti-AQP2 antibodies, and then stained with secondary tetramethyl rhodamine goat anti-mouse (1:500) and AF-488-conjugated goat anti-rabbit (1:500) antibodies, respectively. WT, wild-type.
    Figure Legend Snippet: Characterization of synapse-associated protein-97 (SAP97) and aquaporin-2 (AQP2) interactions in LLC-PK 1 cells and in mouse kidneys. A and B : coimmunoprecipitations (co-IPs) between c-Myc-AQP2 and SAP97. A : anti-c-Myc resin ( lane 2 ) but not preimmune IgG ( lane 3 ) coimmunoprecipitated SAP97 from c-Myc-AQP2-expressing cells. B : anti-SAP97 IgG resin coimmunoprecipitated AQP2 from c-Myc-AQP2-expressing cells. C : co-IPs between AQP2∆PDZ and SAP97. Inputs ( lane 1 ) represent ~4% of total cell lysate from each condition. D : LLC-PK 1 cells expressing c-Myc-WT AQP2 (pseudo red) and SAP97-green fluorescent protein (GFP) (pseudo green) were fixed, permeabilized, stained with CF555 anti-c-Myc IgG, and visualized by dual confocal microscopy. E : medullary slices from C57 black mouse kidneys were fixed, incubated with monoclonal anti-SAP97 and rabbit anti-AQP2 antibodies, and then stained with secondary tetramethyl rhodamine goat anti-mouse (1:500) and AF-488-conjugated goat anti-rabbit (1:500) antibodies, respectively. WT, wild-type.

    Techniques Used: Expressing, Staining, Confocal Microscopy, Incubation

    32) Product Images from "Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus ▿ ▿ †"

    Article Title: Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus ▿ ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01664-07

    Oligomerization of CVYV P1b in solution. N-terminally tagged CVYV P1b proteins purified by affinity chromatography were analyzed by gel filtration-FPLC. (A) Elution fractions of NTAP-P1b, intact or digested with acTEV protease (NCBP-P1b), were subjected to Western blot analysis with PAP complex or biotinylated calmodulin, respectively. (B) Elution fractions of NTAP-P1b mutants were subjected to Western blot analysis with PAP complex. Arrows indicate the elution positions of the following molecular mass markers: aldolase (158 kDa), serum albumin (68 kDa), and ovalbumin (50 kDa).
    Figure Legend Snippet: Oligomerization of CVYV P1b in solution. N-terminally tagged CVYV P1b proteins purified by affinity chromatography were analyzed by gel filtration-FPLC. (A) Elution fractions of NTAP-P1b, intact or digested with acTEV protease (NCBP-P1b), were subjected to Western blot analysis with PAP complex or biotinylated calmodulin, respectively. (B) Elution fractions of NTAP-P1b mutants were subjected to Western blot analysis with PAP complex. Arrows indicate the elution positions of the following molecular mass markers: aldolase (158 kDa), serum albumin (68 kDa), and ovalbumin (50 kDa).

    Techniques Used: Purification, Affinity Chromatography, Filtration, Fast Protein Liquid Chromatography, Western Blot

    33) Product Images from "Anti-HSP70 Autoantibody Formation by B-1 Cells in Toxoplasma gondii-Infected Mice"

    Article Title: Anti-HSP70 Autoantibody Formation by B-1 Cells in Toxoplasma gondii-Infected Mice

    Journal: Infection and Immunity

    doi:

    (A and B) Anti-mHSP70 autoantibodies as well as anti-TgHSP70 antibodies were produced in T. gondii -infected BALB/c and B6 mice. BALB/c and B6 mice were perorally infected with five T. gondii cysts of the Fukaya strain. By using rmHSP70 and rTgHSP70 protein as antigens, formation of anti-mHSP70 autoantibody (A) and anti-TgHSP70 antibody (B) in the sera of infected BALB/c and B6 mice was tested weekly by ELISA. (C and D) Isotype specificity of anti-mHSP70 autoantibody generated by T. gondii -infected mice. BALB/c and B6 mice were perorally infected with five T. gondii cysts of the Fukaya strain. Production of anti-mHSP70 IgG autoantibodies (C) or anti-mHSP70 IgM autoantibodies (D) in the sera of T. gondii -infected BALB/c and B6 mice was tested weekly by ELISA using alkaline phosphatase-conjugated anti-mouse IgG antibody or anti-mouse IgM antibody as the secondary antibody. (E and F) Titration analysis of anti-mHSP70 autoantibody and anti-TgHSP70 antibody in the sera of T. gondii -infected BALB/c and B6 mice. After dilution of the sera of BALB/c and B6 mice 5 weeks after T. gondii infection as described in Materials and Methods, the titration of anti-mHSP70 autoantibody (E) and anti-TgHSP70 antibody (F) was analyzed by ELISA. Symbols: □, anti-mHSP70 autoantibodies in BALB/c mice; ■, anti-mHSP70 autoantibodies in B6 mice; ○, anti-TgHSP70 antibodies in BALB/c mice; ●, anti-TgHSP70 antibodies in B6 mice. ∗, P
    Figure Legend Snippet: (A and B) Anti-mHSP70 autoantibodies as well as anti-TgHSP70 antibodies were produced in T. gondii -infected BALB/c and B6 mice. BALB/c and B6 mice were perorally infected with five T. gondii cysts of the Fukaya strain. By using rmHSP70 and rTgHSP70 protein as antigens, formation of anti-mHSP70 autoantibody (A) and anti-TgHSP70 antibody (B) in the sera of infected BALB/c and B6 mice was tested weekly by ELISA. (C and D) Isotype specificity of anti-mHSP70 autoantibody generated by T. gondii -infected mice. BALB/c and B6 mice were perorally infected with five T. gondii cysts of the Fukaya strain. Production of anti-mHSP70 IgG autoantibodies (C) or anti-mHSP70 IgM autoantibodies (D) in the sera of T. gondii -infected BALB/c and B6 mice was tested weekly by ELISA using alkaline phosphatase-conjugated anti-mouse IgG antibody or anti-mouse IgM antibody as the secondary antibody. (E and F) Titration analysis of anti-mHSP70 autoantibody and anti-TgHSP70 antibody in the sera of T. gondii -infected BALB/c and B6 mice. After dilution of the sera of BALB/c and B6 mice 5 weeks after T. gondii infection as described in Materials and Methods, the titration of anti-mHSP70 autoantibody (E) and anti-TgHSP70 antibody (F) was analyzed by ELISA. Symbols: □, anti-mHSP70 autoantibodies in BALB/c mice; ■, anti-mHSP70 autoantibodies in B6 mice; ○, anti-TgHSP70 antibodies in BALB/c mice; ●, anti-TgHSP70 antibodies in B6 mice. ∗, P

    Techniques Used: Produced, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Generated, Titration

    34) Product Images from "Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells"

    Article Title: Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M033639

    Components of the ERAD pathway required for proteasomal degradation of hamster HMG-CoA reductase in  Drosophila  S2 cells. S2 cells were set up on day 0 in 6-well plates at a density of 1 × 10 6  cells per well in medium B. Immediately after plating, cells were incubated with 15 μg of dsRNA targeted against the indicated endogenous mRNAs. Following incubation for 1 h, the cells received 2 ml of medium C supplemented with 10% HI-LPDS. On day 1, cells were washed and transfected in medium B with 0.5 μg of pAc-HMG-Red-T7 (TM1-8) and 0.1 μg pAc-Insig-1-Myc in medium B using Maxfect™ Transfection Reagent as described in Materials and Methods. On day 2, each well received 1 ml of medium B supplemented with 20% HI-LPDS. Cells were treated on day 3 with medium C supplemented with 10% HI-LPDS in the absence or presence of 2.5 μM 25-HC plus 10 mM mevalonate (Mev.). Following incubation for 4 h, cells were harvested and lysed in detergent-containing buffer; aliquots of the resulting lysates (50 μg of protein/lane) were separated by 10% SDS-PAGE, the proteins were transferred to nitrocellulose membranes, followed by immunoblot analysis with anti-T7 IgG (against reductase), IgG-9E10 (against Insig-1), and anti-E1 IgG. The numbers to the side of immunoblots are referred to as “panels” in the text. Immunoblots shown in panels 4–6 were cropped from the same gel exposed to film for an identical period of time.
    Figure Legend Snippet: Components of the ERAD pathway required for proteasomal degradation of hamster HMG-CoA reductase in Drosophila S2 cells. S2 cells were set up on day 0 in 6-well plates at a density of 1 × 10 6 cells per well in medium B. Immediately after plating, cells were incubated with 15 μg of dsRNA targeted against the indicated endogenous mRNAs. Following incubation for 1 h, the cells received 2 ml of medium C supplemented with 10% HI-LPDS. On day 1, cells were washed and transfected in medium B with 0.5 μg of pAc-HMG-Red-T7 (TM1-8) and 0.1 μg pAc-Insig-1-Myc in medium B using Maxfect™ Transfection Reagent as described in Materials and Methods. On day 2, each well received 1 ml of medium B supplemented with 20% HI-LPDS. Cells were treated on day 3 with medium C supplemented with 10% HI-LPDS in the absence or presence of 2.5 μM 25-HC plus 10 mM mevalonate (Mev.). Following incubation for 4 h, cells were harvested and lysed in detergent-containing buffer; aliquots of the resulting lysates (50 μg of protein/lane) were separated by 10% SDS-PAGE, the proteins were transferred to nitrocellulose membranes, followed by immunoblot analysis with anti-T7 IgG (against reductase), IgG-9E10 (against Insig-1), and anti-E1 IgG. The numbers to the side of immunoblots are referred to as “panels” in the text. Immunoblots shown in panels 4–6 were cropped from the same gel exposed to film for an identical period of time.

    Techniques Used: Incubation, Transfection, SDS Page, Western Blot

    35) Product Images from "Subclinical Prion Disease Induced by Oral Inoculation"

    Article Title: Subclinical Prion Disease Induced by Oral Inoculation

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.14.7991-7998.2003

    Detection of PrP Sc in brain tissue of wild-type mice with subclinical prion disease orally inoculated with RML 5.0 prions. Aliquots of 10% (wt/vol) whole-brain homogenates (wild-type mice) or brain stems ( tga20 mice) from mice that had succumbed to terminal prion disease (i.c. or i.p. inoculated) or orally inoculated mice that were euthanized either 375 or 525 days after prion inoculation were treated with PK and subjected to Western blotting with rabbit polyclonal anti-PrP serum XN and enhanced chemiluminescence. Sample portions (10 μl for non-PK-treated samples and 15 μl for PK-treated samples) (equivalent to 40 to 50 μg of total protein) were loaded on the gel. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. Samples were digested without (−) or with (+) PK.
    Figure Legend Snippet: Detection of PrP Sc in brain tissue of wild-type mice with subclinical prion disease orally inoculated with RML 5.0 prions. Aliquots of 10% (wt/vol) whole-brain homogenates (wild-type mice) or brain stems ( tga20 mice) from mice that had succumbed to terminal prion disease (i.c. or i.p. inoculated) or orally inoculated mice that were euthanized either 375 or 525 days after prion inoculation were treated with PK and subjected to Western blotting with rabbit polyclonal anti-PrP serum XN and enhanced chemiluminescence. Sample portions (10 μl for non-PK-treated samples and 15 μl for PK-treated samples) (equivalent to 40 to 50 μg of total protein) were loaded on the gel. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. Samples were digested without (−) or with (+) PK.

    Techniques Used: Mouse Assay, Western Blot

    36) Product Images from "Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1"

    Article Title: Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.23.12888-12900.2004

    Identification of mouse Sur2 as a specific MAV-1 E1A-interacting protein. (A) Large-scale GST-mE1A pulldown assay. Lane 1, protein molecular size standards; lanes 2 to 4, purified GST, GST-mE1A, and GST-Cter1 fusion proteins, respectively, were bound to glutathione-Sepharose beads without mixing with any mammalian cell nuclear extracts (−); lanes 5 to 7, nuclear extracts from 5 × 10 8  mouse MBMECs were precleared sequentially against glutathione-Sepharose beads and GST beads, and then an equal amount of the precleared mammalian nuclear extracts was added (+) to GST beads, GST-mE1A beads, and GST-Cter1 beads, respectively. Beads were washed, and the bound proteins were eluted off the beads by boiling in protein loading buffer. Proteins binding specifically to GST-mE1A were identified by comparing lane 6 with lanes 3 and 5. The arrowhead shows Sur2 protein. An independent duplicate analysis of both MBMEC and 3T6 cell lines gave similar results, with identification of Sur2 (data not shown). (B) Sur2 protein interacts with GST-mE1A (full-length E1A) in GST pulldowns. GST pulldown assays were performed by incubating the nuclear extracts from 3T6 cells (lanes 1 to 3) or MBMECs (lanes 4 to 6) with GST beads, GST-Cter1 beads, or GST-mE1A beads, as indicated. Monoclonal antibody against Sur2 (BD Pharmingen) (1:1,000) was used for Western blots. Whole-cell lysates (WCL) were used as a positive control (lane 8). Lane 7, protein molecular size standards. The arrowhead shows the Sur2 position. (C) MAV-1 E1A protein interacts with Sur2 in virus-infected cells. MBMECs were mock or MAV-1 infected at an MOI of 5 and harvested at 40 h postinfection. Normal rabbit serum or AKO7-147 (E1A) (both purified by DEAE Affi-Gel blue chromatography) was mixed with whole-cell lysates of MBMECs to carry out the immunoprecipitation. The immunoprecipitates were electrophoresed on 8% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. Monoclonal antibody against Sur2 was used in Western blots. The arrowhead shows the Sur2 position.
    Figure Legend Snippet: Identification of mouse Sur2 as a specific MAV-1 E1A-interacting protein. (A) Large-scale GST-mE1A pulldown assay. Lane 1, protein molecular size standards; lanes 2 to 4, purified GST, GST-mE1A, and GST-Cter1 fusion proteins, respectively, were bound to glutathione-Sepharose beads without mixing with any mammalian cell nuclear extracts (−); lanes 5 to 7, nuclear extracts from 5 × 10 8 mouse MBMECs were precleared sequentially against glutathione-Sepharose beads and GST beads, and then an equal amount of the precleared mammalian nuclear extracts was added (+) to GST beads, GST-mE1A beads, and GST-Cter1 beads, respectively. Beads were washed, and the bound proteins were eluted off the beads by boiling in protein loading buffer. Proteins binding specifically to GST-mE1A were identified by comparing lane 6 with lanes 3 and 5. The arrowhead shows Sur2 protein. An independent duplicate analysis of both MBMEC and 3T6 cell lines gave similar results, with identification of Sur2 (data not shown). (B) Sur2 protein interacts with GST-mE1A (full-length E1A) in GST pulldowns. GST pulldown assays were performed by incubating the nuclear extracts from 3T6 cells (lanes 1 to 3) or MBMECs (lanes 4 to 6) with GST beads, GST-Cter1 beads, or GST-mE1A beads, as indicated. Monoclonal antibody against Sur2 (BD Pharmingen) (1:1,000) was used for Western blots. Whole-cell lysates (WCL) were used as a positive control (lane 8). Lane 7, protein molecular size standards. The arrowhead shows the Sur2 position. (C) MAV-1 E1A protein interacts with Sur2 in virus-infected cells. MBMECs were mock or MAV-1 infected at an MOI of 5 and harvested at 40 h postinfection. Normal rabbit serum or AKO7-147 (E1A) (both purified by DEAE Affi-Gel blue chromatography) was mixed with whole-cell lysates of MBMECs to carry out the immunoprecipitation. The immunoprecipitates were electrophoresed on 8% polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membranes. Monoclonal antibody against Sur2 was used in Western blots. The arrowhead shows the Sur2 position.

    Techniques Used: Purification, Binding Assay, Western Blot, Positive Control, Infection, Chromatography, Immunoprecipitation

    37) Product Images from "Virulence Role of V Antigen of Yersinia pestis at the Bacterial Surface"

    Article Title: Virulence Role of V Antigen of Yersinia pestis at the Bacterial Surface

    Journal: Infection and Immunity

    doi:

    LcrV-specific antibody does not neutralize Yop-targeting activity by nonpreinduced Y. pestis . (A) α-HTV or α-YopM antibody was added at 175 μg/ml to warm RPMI containing Y. pestis KIM8-3002, and the mixture was added without centrifugation to monolayers of HeLa cells at an MOI of 10. After incubation for 3 h at 37°C with 5% CO 2 , the cultures were visualized by phase-contrast microscopy and photographed through a green filter. (B) HeLa cell monolayers were untreated (UI) (lane 1) or infected with Y. pestis KIM8-3002 at an MOI of 10 in the presence (lanes 6 to 9) or absence (lanes 2 to 5) of LcrV-specific Fab antibody fragments (α-HTV Fab). For this experiment, Fab antibody fragments were present at 26.5 μg/ml, corresponding to a 1,000-fold molar excess over estimated LcrV levels. After 4 h, replicate wells were treated with trypsin at 100 μg/ml prior to harvesting (lanes 4, 5, 8, and 9) or were harvested directly. Samples were fractionated into cell-free supernatants (lanes 3, 5, 7, and 9) and HeLa cell soluble fractions (lanes 1, 2, 4, 6, and 8). To verify that released LcrV could be quantitatively bound by the Fab fragments, LcrV bound by α-HTV Fab was immunoprecipitated (Immunoppt) from a nontrypsinized supernatant fraction by using anti-IgG and protein A-agarose beads. The combined void and protein A column washes (FT), elution (E) fractions, and fractionated culture samples were analyzed by immunoblotting with α-HTV and α-YopE. Preparations of whole antibody (IgG) (lane 12) and Fab fragments (lane 13) were also resolved as references. Proteins were visualized by probing with horseradish peroxidase-coupled secondary antibody, developed with ECL reagent, and exposed to film.
    Figure Legend Snippet: LcrV-specific antibody does not neutralize Yop-targeting activity by nonpreinduced Y. pestis . (A) α-HTV or α-YopM antibody was added at 175 μg/ml to warm RPMI containing Y. pestis KIM8-3002, and the mixture was added without centrifugation to monolayers of HeLa cells at an MOI of 10. After incubation for 3 h at 37°C with 5% CO 2 , the cultures were visualized by phase-contrast microscopy and photographed through a green filter. (B) HeLa cell monolayers were untreated (UI) (lane 1) or infected with Y. pestis KIM8-3002 at an MOI of 10 in the presence (lanes 6 to 9) or absence (lanes 2 to 5) of LcrV-specific Fab antibody fragments (α-HTV Fab). For this experiment, Fab antibody fragments were present at 26.5 μg/ml, corresponding to a 1,000-fold molar excess over estimated LcrV levels. After 4 h, replicate wells were treated with trypsin at 100 μg/ml prior to harvesting (lanes 4, 5, 8, and 9) or were harvested directly. Samples were fractionated into cell-free supernatants (lanes 3, 5, 7, and 9) and HeLa cell soluble fractions (lanes 1, 2, 4, 6, and 8). To verify that released LcrV could be quantitatively bound by the Fab fragments, LcrV bound by α-HTV Fab was immunoprecipitated (Immunoppt) from a nontrypsinized supernatant fraction by using anti-IgG and protein A-agarose beads. The combined void and protein A column washes (FT), elution (E) fractions, and fractionated culture samples were analyzed by immunoblotting with α-HTV and α-YopE. Preparations of whole antibody (IgG) (lane 12) and Fab fragments (lane 13) were also resolved as references. Proteins were visualized by probing with horseradish peroxidase-coupled secondary antibody, developed with ECL reagent, and exposed to film.

    Techniques Used: Activity Assay, Centrifugation, Incubation, Microscopy, Infection, Immunoprecipitation

    38) Product Images from "Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain"

    Article Title: Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179615

    Co-localization of Trypanosoma cruzi AP-1 complex γ subunit (TcAP1-γ) with T . cruzi Golgi markers. Epimastigotes expressing the tagged Golgi marker Huntingtin interacting protein TcHIP-A/C (A-D) or tagged clathrin light chain TcCLC-A/C (E-H) were labelled with an anti-TcAP1-γ mAb 211.F7 that was detected with anti-mouse IgG conjugated to Alexa Fluor 488 followed by incubation with rabbit IgG anti-protein A for TcHIP-A/C and TcCLC-A/C, that were detected with anti-rabbit IgG conjugated to Alexa Fluor 546. Nuclear (n) and kinetoplast (k) DNA were stained with Hoechst 33342. Arrows indicate co-localization. DIC, differential interference contrast microscopy. Scale bar = 5μm.
    Figure Legend Snippet: Co-localization of Trypanosoma cruzi AP-1 complex γ subunit (TcAP1-γ) with T . cruzi Golgi markers. Epimastigotes expressing the tagged Golgi marker Huntingtin interacting protein TcHIP-A/C (A-D) or tagged clathrin light chain TcCLC-A/C (E-H) were labelled with an anti-TcAP1-γ mAb 211.F7 that was detected with anti-mouse IgG conjugated to Alexa Fluor 488 followed by incubation with rabbit IgG anti-protein A for TcHIP-A/C and TcCLC-A/C, that were detected with anti-rabbit IgG conjugated to Alexa Fluor 546. Nuclear (n) and kinetoplast (k) DNA were stained with Hoechst 33342. Arrows indicate co-localization. DIC, differential interference contrast microscopy. Scale bar = 5μm.

    Techniques Used: Expressing, Marker, Incubation, Staining, Microscopy

    39) Product Images from "A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis"

    Article Title: A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2000.00143.x

    Cell counts of infiltrating inflammatory cells in the bronchoalveolar lavage (BAL) of adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) mice treated with tumour necrosis factor-α (TNF-α)-neutralizing antibody. Blinded differential counts were performed on Wright–Giemsa-stained BAL cytospins. (a) The bar graph shows the mean number (±SEM) of infiltrating lymphocytes and monocytes/immature dendritic cells in the BAL (ordinate) of experimental mice (groups A, A′ and B [abscissa]) 3 days after intratracheal (i.t.) challenge with 2,4,6-trinitrobenzene sulphonic acid (TNBS). The T cells in the BAL samples were identified by flow cytometry of fluorescein isothiocyanate (FITC)-labelled CD3 + BAL cells. (b) The bar graph shows the mean number of CD3 + T cells in the BAL of experimental mice (A, A′ and B) 3 days after i.t. challenge. Five mice were used in each experimental group. An asterisk (*) indicates a statistically significant difference ( P ≤0·05) between groups A and A′ or groups A and B.
    Figure Legend Snippet: Cell counts of infiltrating inflammatory cells in the bronchoalveolar lavage (BAL) of adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) mice treated with tumour necrosis factor-α (TNF-α)-neutralizing antibody. Blinded differential counts were performed on Wright–Giemsa-stained BAL cytospins. (a) The bar graph shows the mean number (±SEM) of infiltrating lymphocytes and monocytes/immature dendritic cells in the BAL (ordinate) of experimental mice (groups A, A′ and B [abscissa]) 3 days after intratracheal (i.t.) challenge with 2,4,6-trinitrobenzene sulphonic acid (TNBS). The T cells in the BAL samples were identified by flow cytometry of fluorescein isothiocyanate (FITC)-labelled CD3 + BAL cells. (b) The bar graph shows the mean number of CD3 + T cells in the BAL of experimental mice (A, A′ and B) 3 days after i.t. challenge. Five mice were used in each experimental group. An asterisk (*) indicates a statistically significant difference ( P ≤0·05) between groups A and A′ or groups A and B.

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

    (a) Flow cytometric analysis of the Mac-1 + /Gr-1 dim population in peripheral blood after intravenous (i.v.) treatment with dichloromethylene diphosphonate (Cl 2 MDP) liposomes. The two-parameter histograms compare the percentage of Mac-1 + /Gr-1 dim cells in peripheral blood from naïve mice that were treated with or without Cl 2 MDP liposomes (i.v., 200 µl/mouse). The blood was collected 2 days after the treatment and stained with phycoerythrin (PE)-conjugated Mac-1 antibody and biotin-conjugated Gr-1 antibody plus fluorescein isothiocyanate (FITC)-conjugated ExtrAvidin®. The samples were analysed using flow cytometry. Peripheral blood monocytes were identified by their Mac-1 + /Gr-1 dim staining pattern and gated on the histograms. The percentage of Mac-1 + /Gr-1 dim cells is indicted in each gate by the rectangle. (b) Differential cell counts of alveolar macrophages (AM) in bronchoalveolar lavage (BAL) following intranasal (i.n.) or intratracheal (i.t.) Cl 2 MDP liposome treatment. The histogram shows the number of AMs in each lung (ordinate) after the naïve mice were treated or not treated with Cl 2 MDP liposomes (abscissa). One dose of 100 µl of Cl 2 MDP liposomes was given i.t. per naïve mouse. Three doses of 33 µl (total 99 µl) of Cl 2 MDP liposomes were given i.n. per naïve mouse during the course of 1 day. BAL was collected 2 days after i.t. or i.n. treatment. BAL cytospins were stained with Wright–Giemsa and evaluated by light microscopy (×40 Nikon). Three mice were used in each group. AMs were identified and counted by differential cell counts (see the Materials and Methods). The number of AMs in each lung was calculated by multiplying the total number of cells in the BAL preparation with the percentage of AMs from differential cell counts. The result is presented as mean±SEM. (c) Hydroxyproline deposition in the lungs of adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) mice treated with Cl 2 MDP liposomes (i.n.). Recipient mice (groups A′, B′ and C′) were treated with Cl 2 MDP liposomes (33 µl/dose×three doses) and irradiated 1 day prior to receiving adoptive transfer of sensitized or non-sensitized cells. The mice in groups A, B and C were treated with phosphate-buffered saline (PBS) liposomes. One day before i.t. challenge, the mice in groups A and A′ received 3×10 7 2,4,6-trinitrobenzene sulphonic acid (TNBS)-sensitized spleen and draining lymph node (inguinal, brachial, axillary) cells; mice in groups B and B′ received 3×10 7 naïve spleen cells, and lymph node (inguinal, brachial, axillary) cells. Group C mice were untreated. Fourteen days after i.t. challenge, the lungs from experimental mice (five mice per group) were harvested and analysed for their hydroxyproline level. The histogram shows the change (Δ) in hydroxyproline level (experimental hydroxyproline – baseline hydroxyproline) in each experimental group (mean±SEM). The baseline hydroxyproline concentration was 20 µg/lung and was calculated as the mean hydroxyproline concentration from five naïve mice that were treated with PBS liposomes (i.n.). An asterisk (*) indicates a statistically significant difference ( P =0·05) between indicated groups. The experiment was repeated twice.
    Figure Legend Snippet: (a) Flow cytometric analysis of the Mac-1 + /Gr-1 dim population in peripheral blood after intravenous (i.v.) treatment with dichloromethylene diphosphonate (Cl 2 MDP) liposomes. The two-parameter histograms compare the percentage of Mac-1 + /Gr-1 dim cells in peripheral blood from naïve mice that were treated with or without Cl 2 MDP liposomes (i.v., 200 µl/mouse). The blood was collected 2 days after the treatment and stained with phycoerythrin (PE)-conjugated Mac-1 antibody and biotin-conjugated Gr-1 antibody plus fluorescein isothiocyanate (FITC)-conjugated ExtrAvidin®. The samples were analysed using flow cytometry. Peripheral blood monocytes were identified by their Mac-1 + /Gr-1 dim staining pattern and gated on the histograms. The percentage of Mac-1 + /Gr-1 dim cells is indicted in each gate by the rectangle. (b) Differential cell counts of alveolar macrophages (AM) in bronchoalveolar lavage (BAL) following intranasal (i.n.) or intratracheal (i.t.) Cl 2 MDP liposome treatment. The histogram shows the number of AMs in each lung (ordinate) after the naïve mice were treated or not treated with Cl 2 MDP liposomes (abscissa). One dose of 100 µl of Cl 2 MDP liposomes was given i.t. per naïve mouse. Three doses of 33 µl (total 99 µl) of Cl 2 MDP liposomes were given i.n. per naïve mouse during the course of 1 day. BAL was collected 2 days after i.t. or i.n. treatment. BAL cytospins were stained with Wright–Giemsa and evaluated by light microscopy (×40 Nikon). Three mice were used in each group. AMs were identified and counted by differential cell counts (see the Materials and Methods). The number of AMs in each lung was calculated by multiplying the total number of cells in the BAL preparation with the percentage of AMs from differential cell counts. The result is presented as mean±SEM. (c) Hydroxyproline deposition in the lungs of adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) mice treated with Cl 2 MDP liposomes (i.n.). Recipient mice (groups A′, B′ and C′) were treated with Cl 2 MDP liposomes (33 µl/dose×three doses) and irradiated 1 day prior to receiving adoptive transfer of sensitized or non-sensitized cells. The mice in groups A, B and C were treated with phosphate-buffered saline (PBS) liposomes. One day before i.t. challenge, the mice in groups A and A′ received 3×10 7 2,4,6-trinitrobenzene sulphonic acid (TNBS)-sensitized spleen and draining lymph node (inguinal, brachial, axillary) cells; mice in groups B and B′ received 3×10 7 naïve spleen cells, and lymph node (inguinal, brachial, axillary) cells. Group C mice were untreated. Fourteen days after i.t. challenge, the lungs from experimental mice (five mice per group) were harvested and analysed for their hydroxyproline level. The histogram shows the change (Δ) in hydroxyproline level (experimental hydroxyproline – baseline hydroxyproline) in each experimental group (mean±SEM). The baseline hydroxyproline concentration was 20 µg/lung and was calculated as the mean hydroxyproline concentration from five naïve mice that were treated with PBS liposomes (i.n.). An asterisk (*) indicates a statistically significant difference ( P =0·05) between indicated groups. The experiment was repeated twice.

    Techniques Used: Flow Cytometry, Mouse Assay, Staining, Cytometry, Affinity Magnetic Separation, Light Microscopy, Adoptive Transfer Assay, Irradiation, Concentration Assay

    Flow cytometric dot-plot analyses of CD3 + and interleukin-12 receptor-positive (IL-12R + ) lymphocytes in adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) lungs treated with dichloromethylene diphosphonate (Cl 2 MDP) liposomes intranasally (i.n.). Recipient mice were treated i.n. with Cl 2 MDP liposomes (33 µl/dose×three doses) or control liposomes 2 days prior to i.t. challenge and received sensitized cells 1 day before i.t. challenge. One day after i.t. challenge, bronchoalveolar lavage (BAL) was collected, stained for surface expression of CD3 and IL-12R (see Materials and methods) and then analysed using flow cytometry. (a) Mice were treated with control liposomes. (b) Mice were treated with Cl 2 MDP liposomes. The percentage of the population that was gated (arrow) is indicated in the forward scatter (FS)/side scatter (SS) dot-plot in (a) and (b) of each panel. To the right of each panel are dot-plots showing CD3 and IL-12R expression on gated cells. The percentage of CD3 + and IL-12R + cells is indicated in each quadrant. A minimum of three mice was used in each experimental group. The experiment was repeated twice. FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: Flow cytometric dot-plot analyses of CD3 + and interleukin-12 receptor-positive (IL-12R + ) lymphocytes in adoptive transfer-hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) lungs treated with dichloromethylene diphosphonate (Cl 2 MDP) liposomes intranasally (i.n.). Recipient mice were treated i.n. with Cl 2 MDP liposomes (33 µl/dose×three doses) or control liposomes 2 days prior to i.t. challenge and received sensitized cells 1 day before i.t. challenge. One day after i.t. challenge, bronchoalveolar lavage (BAL) was collected, stained for surface expression of CD3 and IL-12R (see Materials and methods) and then analysed using flow cytometry. (a) Mice were treated with control liposomes. (b) Mice were treated with Cl 2 MDP liposomes. The percentage of the population that was gated (arrow) is indicated in the forward scatter (FS)/side scatter (SS) dot-plot in (a) and (b) of each panel. To the right of each panel are dot-plots showing CD3 and IL-12R expression on gated cells. The percentage of CD3 + and IL-12R + cells is indicated in each quadrant. A minimum of three mice was used in each experimental group. The experiment was repeated twice. FITC, fluorescein isothiocyanate.

    Techniques Used: Flow Cytometry, Adoptive Transfer Assay, Mouse Assay, Staining, Expressing, Cytometry

    40) Product Images from "Internalization of novel non-viral vector TAT-streptavidin into human cells"

    Article Title: Internalization of novel non-viral vector TAT-streptavidin into human cells

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-1

    Quantification of internalized TAT-SA in living HeLa cells . (A) Quantitative analysis of the colocalization between both TAT-SA-A488 and TRITC-labeled transferrin and TRITC-labeled dextran (10 kD) at 15 min post transduction. (B) The relative fluorescence intensity of internalized TAT-SA-A488 was measured in cells treated with cytochalasin D (cytD), amiloride (ami) or nocodazole (noco) at 4 h post transduction. Control cells (C) were untreated. The fluorescence intensity data was collected from multiple series of cells by confocal microcopy and processed with the 3D LSM program.
    Figure Legend Snippet: Quantification of internalized TAT-SA in living HeLa cells . (A) Quantitative analysis of the colocalization between both TAT-SA-A488 and TRITC-labeled transferrin and TRITC-labeled dextran (10 kD) at 15 min post transduction. (B) The relative fluorescence intensity of internalized TAT-SA-A488 was measured in cells treated with cytochalasin D (cytD), amiloride (ami) or nocodazole (noco) at 4 h post transduction. Control cells (C) were untreated. The fluorescence intensity data was collected from multiple series of cells by confocal microcopy and processed with the 3D LSM program.

    Techniques Used: Labeling, Transduction, Fluorescence

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