alexa 488 conjugated anti rabbit igg secondary antibodies  (Jackson Immuno)

 
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    Name:
    Alexa Fluor 488 AffiniPure Goat Anti Rabbit IgG F ab 2 fragment specific
    Description:
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with the F ab 2 Fab portion of rabbit IgG It also reacts with the light chains of other rabbit immunoglobulins No antibody was detected against the Fc portion of rabbit IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    Catalog Number:
    111-545-006
    Price:
    154.0
    Category:
    Whole IgG Affinity Purified Antibodies
    Conjugate:
    Alexa Fluor 488
    Size:
    1 5 mg
    Format:
    Whole IgG
    Host:
    Goat
    Buy from Supplier


    Structured Review

    Jackson Immuno alexa 488 conjugated anti rabbit igg secondary antibodies
    Secretion of Type I collagen was induced by purified <t>IgG</t> from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P
    Whole IgG antibodies are isolated as intact molecules from antisera by immunoaffinity chromatography They have an Fc portion and two antigen binding Fab portions joined together by disulfide bonds and therefore they are divalent The average molecular weight is reported to be about 160 kDa The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective Based on immunoelectrophoresis and or ELISA the antibody reacts with the F ab 2 Fab portion of rabbit IgG It also reacts with the light chains of other rabbit immunoglobulins No antibody was detected against the Fc portion of rabbit IgG or against non immunoglobulin serum proteins The antibody may cross react with immunogloublins from other species
    https://www.bioz.com/result/alexa 488 conjugated anti rabbit igg secondary antibodies/product/Jackson Immuno
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa 488 conjugated anti rabbit igg secondary antibodies - by Bioz Stars, 2021-07
    93/100 stars

    Images

    1) Product Images from "Alteration of fibroblast phenotype by asbestos-induced autoantibodies"

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies

    Journal: Journal of immunotoxicology

    doi: 10.3109/1547691X.2011.562257

    Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P
    Figure Legend Snippet: Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P

    Techniques Used: Purification, Mouse Assay, Fluorescence

    Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P
    Figure Legend Snippet: Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P

    Techniques Used: Binding Assay, Mouse Assay, Staining, Incubation, In-Cell ELISA

    Related Articles

    Incubation:

    Article Title: Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease
    Article Snippet: Primary antibodies were used at the following dilutions: rabbit anti–glutamine synthetase (anti-GS) IgG at 1:3000 and rat 1D4B anti-lysosome-associated membrane glycoprotein 1 (Lamp1) IgG2a supernatant (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) at 1:50. .. After washing cells with ice-cold PBSBS 4 times for 5 min each, cells were incubated with secondary antibodies and Hoechst 33342 at 4°C for 1 h. Secondary antibodies and Hoechst 33342 were at the following dilutions: Alexa Fluor 488–conjugated goat anti-rabbit IgG at 1:500, Cy3-conjugated goat anti-rabbit IgG (1.5 mg/ml; Jackson ImmunoResearch Laboratories) at 1:330, Cy3-conjugated goat anti-rat IgG at 1:330, and Hoechst 33342 at 1:813. .. After washing the cells with PBSBS, they were incubated with 0.2 U/ml of Alexa Fluor 488–conjugated phalloidin (Thermo Fisher Scientific) in PBSB for 20 min at 4°C to label the apical microvilli of the Müller glia ( ).

    Article Title: CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery
    Article Snippet: Samples were incubated with primary antibody (1:400 rabbit anti-mouse Tie-2, Santa Cruz Biotechnology, H-176, sc-9026) and 1:50 anti-CD36 (BD PharMingen) overnight at 4°C; for some samples the primary antibodies were omitted as controls. .. Tissues were then incubated with conjugated secondary antibody Cy3-conjugated goat anti-mouse (Jackson ImmunoResearch) and AF-488-conjugated goat anti-rabbit (Jackson ImmunoResearch) for 1 h at room temperature. ..

    Article Title: Mediation of Mucosal Immunoglobulins in Buccal Cavity of Teleost in Antibacterial Immunity
    Article Snippet: Subsequently, the paraffin sections of BM were incubated at 4°C overnight with rabbit anti-trout IgT (pAb; 0.5 μg ml–1 ) and mouse anti-trout IgM (IgG1 isotype; 1 μg ml–1 ). .. After washing with PBS, paraffin sections were incubated for 45 min at room temperature with Alexa Fluor 488-conjugated AffiniPure Goat anti-rabbit IgG and Cy3-conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.) at 2.5 μg ml–1 each. .. EdU+ cell detection was performed according to the manufacturer’s instructions (Click-iT EdU Alexa Fluor 647 Imaging Kit, Invitrogen).

    Staining:

    Article Title: Transferrin Receptor 2 Is Frequently and Highly Expressed in Glioblastomas
    Article Snippet: Coverslips were stained with mouse mAbs directed against TfR2 (15 minutes at 37°C), followed by a Texas Red-conjugated goat antimouse IgG (Jackson Immunoresearch, West Grove, PA). .. Cells were then fixed and permeabilized (4% paraformaldehyde/2% sucrose, 10 minutes at room temperature, plus 0.1% saponin, 30 minutes at room temperature), saturated (1% normal goat serum), and stained with a rabbit polyclonal anti-caveolin-1 (BD Bioscience, Bedford, MA), followed by an Alexa 488-conjugated antirabbit IgG (Jackson Immunoresearch). .. All images were acquired using an Olympus 1x70 (FV300 System) confocal microscope and processed with the Analysis software (Olympus Life Sciences, Milan, Italy).

    Article Title: Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-?B activity: a comparative study with the HTLV-1 Tax-1 protein
    Article Snippet: The anti-GM130 was from BD Biosciences. .. The secondary antibodies used for immunofluorescence staining were Dylight 488-conjugated goat anti-mouse IgG2a, Dylight 549- or 488-conjugated goat anti-rabbit IgG and Dylight 649-conjugated goat anti-mouse IgG1 from Jackson ImmunoResearch. .. Luciferase assays Tax-1- and Tax-2B-mediated activation of gene expression via the NF-κB pathway was assayed by dual luciferase assays using the NF-κB-luc reporter construct.

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies
    Article Snippet: Antibodies to PDGF-Rα, vimentin, or TGFβ-RII (R & D Systems) were used as positive controls at appropriate dilutions to yield 1 μg/ml final concentrations per well. .. To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA). ..

    Immunofluorescence:

    Article Title: Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-?B activity: a comparative study with the HTLV-1 Tax-1 protein
    Article Snippet: The anti-GM130 was from BD Biosciences. .. The secondary antibodies used for immunofluorescence staining were Dylight 488-conjugated goat anti-mouse IgG2a, Dylight 549- or 488-conjugated goat anti-rabbit IgG and Dylight 649-conjugated goat anti-mouse IgG1 from Jackson ImmunoResearch. .. Luciferase assays Tax-1- and Tax-2B-mediated activation of gene expression via the NF-κB pathway was assayed by dual luciferase assays using the NF-κB-luc reporter construct.

    Transduction:

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies
    Article Snippet: Antibodies to PDGF-Rα, vimentin, or TGFβ-RII (R & D Systems) were used as positive controls at appropriate dilutions to yield 1 μg/ml final concentrations per well. .. To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA). ..

    Mouse Assay:

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies
    Article Snippet: Antibodies to PDGF-Rα, vimentin, or TGFβ-RII (R & D Systems) were used as positive controls at appropriate dilutions to yield 1 μg/ml final concentrations per well. .. To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA). ..

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  • 93
    Jackson Immuno alexa 488 conjugated anti rabbit igg secondary antibodies
    Secretion of Type I collagen was induced by purified <t>IgG</t> from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P
    Alexa 488 Conjugated Anti Rabbit Igg Secondary Antibodies, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 488 conjugated anti rabbit igg secondary antibodies/product/Jackson Immuno
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa 488 conjugated anti rabbit igg secondary antibodies - by Bioz Stars, 2021-07
    93/100 stars
      Buy from Supplier

    98
    Jackson Immuno alexa fluor 488 conjugated anti rabbit
    Induction of LDLR expression and activity by triciribine. ( a . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured, vehicle-treated cells. ( b . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured cells harvested at 0 hour. ( c ) Sterol-fed HepG2 cells were treated with 1 µM for 14 hours and then examined by fluorescence microscopy as described in Materials and Methods. Green: LDLR, <t>Alexa</t> fluor 488, blue: nuclei, DAPI. One representative experiment is shown (n = 3). ( d ) Sterol-fed HepG2 cells were exposed to vehicle or 1 μM triciribine for 14 hours before analysis for mean fluorescence intensity (as a measure of cell-surface LDLR levels) by flow cytometry. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( e ) Sterol-fed HepG2 cells were cultured and treated as described in d. After exposure of cells to DiD-LDL (10 μg/ml) during the last 2 hours of treatment, cells were harvested and analyzed by flow cytometry to determine the mean fluorescent intensity of internalized DiD-LDL. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( f . Error bars represent SD. * p
    Alexa Fluor 488 Conjugated Anti Rabbit, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated anti rabbit/product/Jackson Immuno
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 conjugated anti rabbit - by Bioz Stars, 2021-07
    98/100 stars
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    95
    Jackson Immuno antirabbit alexa fluor 488 conjugate
    FOX1 localizes to the plasma membrane in iron-deprived Chlamydomonas cells and is oriented with the MCO domain outside the cell. The cell wall mutant strain, cc-425, was grown in TAP medium containing either 10 μM iron for FOX1 repression (A) or no iron plus 100 μM ferrozine for FOX1 induction (B). Cells were plated and left unpermeabilized prior to treatment with anti-FOX1 and <t>Alexa</t> <t>fluor</t> 488 conjugate antibodies. As a control for membrane integrity, permeabilized (C) and unpermeabilized cells (D) were probed for ATP synthase which is localized to the chloroplast. Dim fluorescence is visualized in the unpermeabilized cells (D), which is likely due to background autofluorescence. The bottom panels represent phase-contrast images of the same cells under each condition.
    Antirabbit Alexa Fluor 488 Conjugate, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antirabbit alexa fluor 488 conjugate/product/Jackson Immuno
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antirabbit alexa fluor 488 conjugate - by Bioz Stars, 2021-07
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    94
    Jackson Immuno alexa fluor 647 conjugated anti rabbit
    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, <t>Alexa</t> fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).
    Alexa Fluor 647 Conjugated Anti Rabbit, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647 conjugated anti rabbit/product/Jackson Immuno
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 647 conjugated anti rabbit - by Bioz Stars, 2021-07
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    Image Search Results


    Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P

    Journal: Journal of immunotoxicology

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies

    doi: 10.3109/1547691X.2011.562257

    Figure Lengend Snippet: Secretion of Type I collagen was induced by purified IgG from asbestos-exposed mice. (A) L929 cells were grown to confluence, treated with purified IgG from saline- or tremolite-exposed mice for 3 days, and then collagen production was detected using anti-collagen Type I antibody followed by Alexa 488-conjugated secondary antibody. Fluorescence was quantified by LSC. (B) Collagen production by L929 cells was also measured by Sircol assay, as described in Materials and methods section. Purified IgG = IgG from tremolite-exposed mice, Asb = tremolite asbestos alone at 40 μg/cm 2 , Asb and IgG = combined treatment with both tremolite and purified IgG from tremolite-instilled mice. N = 4 in each group, * P

    Article Snippet: To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA).

    Techniques: Purification, Mouse Assay, Fluorescence

    Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P

    Journal: Journal of immunotoxicology

    Article Title: Alteration of fibroblast phenotype by asbestos-induced autoantibodies

    doi: 10.3109/1547691X.2011.562257

    Figure Lengend Snippet: Binding of serum antibodies to mouse fibroblasts indicates presence of anti-fibroblast antibodies in serum from asbestos-instilled mice. (A) Mouse primary skin fibroblasts were fixed with paraformaldehyde and stained using serum from (left) saline- or (right) tremolite-exposed mice as the primary antibody, followed by incubation with Alexa-488-conjugated anti-mouse IgG secondary Ab. Nuclei are stained with propidium iodide. Images (at 400×) are representative of multiple experiments. (B) A cell-based ELISA was performed using L929 mouse fibroblasts, to measure the binding of serum antibodies to the cells. As in A, binding of serum antibodies was compared between sera from saline- or tremolite-treated mice. N = 7, * P

    Article Snippet: To determine whether the autoantibodies had an effect on signal transduction in fibroblasts, L929 cells were plated to 8-well chamber slides and treated with pooled serum from asbestos-treated mice or pooled cleared serum (IgG removed using Protein G) for 2 h. The cells were then washed, fixed with ice cold acetone, blocked as above, and stained with rabbit antibodies to STAT-1 (H95) or SMAD2/3 (FL-425) (both Santa Cruz Biotech) followed by Alexa 488-conjugated anti-rabbit IgG secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA).

    Techniques: Binding Assay, Mouse Assay, Staining, Incubation, In-Cell ELISA

    Induction of LDLR expression and activity by triciribine. ( a . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured, vehicle-treated cells. ( b . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured cells harvested at 0 hour. ( c ) Sterol-fed HepG2 cells were treated with 1 µM for 14 hours and then examined by fluorescence microscopy as described in Materials and Methods. Green: LDLR, Alexa fluor 488, blue: nuclei, DAPI. One representative experiment is shown (n = 3). ( d ) Sterol-fed HepG2 cells were exposed to vehicle or 1 μM triciribine for 14 hours before analysis for mean fluorescence intensity (as a measure of cell-surface LDLR levels) by flow cytometry. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( e ) Sterol-fed HepG2 cells were cultured and treated as described in d. After exposure of cells to DiD-LDL (10 μg/ml) during the last 2 hours of treatment, cells were harvested and analyzed by flow cytometry to determine the mean fluorescent intensity of internalized DiD-LDL. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( f . Error bars represent SD. * p

    Journal: Scientific Reports

    Article Title: Triciribine increases LDLR expression and LDL uptake through stabilization of LDLR mRNA

    doi: 10.1038/s41598-018-34237-6

    Figure Lengend Snippet: Induction of LDLR expression and activity by triciribine. ( a . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured, vehicle-treated cells. ( b . The graph in the right panel shows quantification of the Western blots, relative to similarly cultured cells harvested at 0 hour. ( c ) Sterol-fed HepG2 cells were treated with 1 µM for 14 hours and then examined by fluorescence microscopy as described in Materials and Methods. Green: LDLR, Alexa fluor 488, blue: nuclei, DAPI. One representative experiment is shown (n = 3). ( d ) Sterol-fed HepG2 cells were exposed to vehicle or 1 μM triciribine for 14 hours before analysis for mean fluorescence intensity (as a measure of cell-surface LDLR levels) by flow cytometry. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( e ) Sterol-fed HepG2 cells were cultured and treated as described in d. After exposure of cells to DiD-LDL (10 μg/ml) during the last 2 hours of treatment, cells were harvested and analyzed by flow cytometry to determine the mean fluorescent intensity of internalized DiD-LDL. The graph shows the results plotted relative to vehicle-treated control (n = 4). ( f . Error bars represent SD. * p

    Article Snippet: Alexa Fluor 488-conjugated anti-rabbit (1:200 dilution; 715–546–150 Jackson ImmunoResearch, Pennsylvania) was used for visualization.

    Techniques: Expressing, Activity Assay, Western Blot, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    FOX1 localizes to the plasma membrane in iron-deprived Chlamydomonas cells and is oriented with the MCO domain outside the cell. The cell wall mutant strain, cc-425, was grown in TAP medium containing either 10 μM iron for FOX1 repression (A) or no iron plus 100 μM ferrozine for FOX1 induction (B). Cells were plated and left unpermeabilized prior to treatment with anti-FOX1 and Alexa fluor 488 conjugate antibodies. As a control for membrane integrity, permeabilized (C) and unpermeabilized cells (D) were probed for ATP synthase which is localized to the chloroplast. Dim fluorescence is visualized in the unpermeabilized cells (D), which is likely due to background autofluorescence. The bottom panels represent phase-contrast images of the same cells under each condition.

    Journal: Eukaryotic Cell

    Article Title: Analysis of the High-Affinity Iron Uptake System at the Chlamydomonas reinhardtii Plasma Membrane ▿

    doi: 10.1128/EC.00310-09

    Figure Lengend Snippet: FOX1 localizes to the plasma membrane in iron-deprived Chlamydomonas cells and is oriented with the MCO domain outside the cell. The cell wall mutant strain, cc-425, was grown in TAP medium containing either 10 μM iron for FOX1 repression (A) or no iron plus 100 μM ferrozine for FOX1 induction (B). Cells were plated and left unpermeabilized prior to treatment with anti-FOX1 and Alexa fluor 488 conjugate antibodies. As a control for membrane integrity, permeabilized (C) and unpermeabilized cells (D) were probed for ATP synthase which is localized to the chloroplast. Dim fluorescence is visualized in the unpermeabilized cells (D), which is likely due to background autofluorescence. The bottom panels represent phase-contrast images of the same cells under each condition.

    Article Snippet: The secondary antibodies used were antirabbit Alexa Fluor 488 conjugate and antimouse Cy5 conjugate (Jackson Immuno Research), both diluted 1:500 in 3% BSA.

    Techniques: Mutagenesis, Fluorescence

    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Journal: Molecules

    Article Title: Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA

    doi: 10.3390/molecules25194505

    Figure Lengend Snippet: TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Article Snippet: Antibodies against LDLR (#3839) (BioVision, Milpitas, CA, USA), β-actin (ab8227) (Abcam, Cambridge, UK) Phospho-LKB1/STK11 (S428)(AF5636) (R & D Systems, Minneapolis, MN, USA), GAPDH (G9295) Sigma-Aldrich, Saint Louis, MO, USA), HuR (ELAVL1)(sc-5261) and Lamin B (sc-6217) (Santa Cruz Biotechnology, Dallas, TX, USA), ERK1/2 (#9102), pERK (#4370) and ZFP36L1/L2 (BRF1/2) (#2119) (Cell Signaling, Beverly, MA, USA), anti-cnot7 (H00029883-A01), (Abnova, Taipei, Taiwan), anti-FLAG (F7425) and anti-HA (#71–5500) (Thermo Fisher Scientific, Waltham, MA, USA).For confocal immunoflouroscence microscopy primary anti-HA (1:100) (#71–5500), with secondary Alexa Fluor 647-conjugated anti-rabbit (715–546–150 Jackson ImmunoResearch, West Grove, PA, USA) (1:200), were used, respectively.

    Techniques: Translocation Assay, Fractionation, Transfection, Immunofluorescence, Microscopy