anti goat immunoglobulin g  (Thermo Fisher)


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  • 99
    Name:
    anti Goat IgG H L Cross Adsorbed
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    Catalog Number:
    sa5-10077
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    Structured Review

    Thermo Fisher anti goat immunoglobulin g
    Assessment of damage to peripheral retinal ganglion cells and optic nerves in the mouse eyes that overexpressed BMP2 in conventional outflow tissues. Thirty-six days after the viral infusion, the mouse eyes and the optic nerves were collected. A : An image from the periphery of the flatmounted retinas of transduced mouse eyes that were immunostained with Brn3a <t>immunoglobulin</t> G (IgG) and imaged with epifluorescence microscopy. B : The morphology of the myelinated axons (toluidine blue stained) of mice 36 days after intracameral transduction with BMP2. The data are representative of images taken of four mice.

    https://www.bioz.com/result/anti goat immunoglobulin g/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti goat immunoglobulin g - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Disease progression in iridocorneal angle tissues of BMP2-induced ocular hypertensive mice with optical coherence tomography"

    Article Title: Disease progression in iridocorneal angle tissues of BMP2-induced ocular hypertensive mice with optical coherence tomography

    Journal: Molecular Vision

    doi:

    Assessment of damage to peripheral retinal ganglion cells and optic nerves in the mouse eyes that overexpressed BMP2 in conventional outflow tissues. Thirty-six days after the viral infusion, the mouse eyes and the optic nerves were collected. A : An image from the periphery of the flatmounted retinas of transduced mouse eyes that were immunostained with Brn3a immunoglobulin G (IgG) and imaged with epifluorescence microscopy. B : The morphology of the myelinated axons (toluidine blue stained) of mice 36 days after intracameral transduction with BMP2. The data are representative of images taken of four mice.
    Figure Legend Snippet: Assessment of damage to peripheral retinal ganglion cells and optic nerves in the mouse eyes that overexpressed BMP2 in conventional outflow tissues. Thirty-six days after the viral infusion, the mouse eyes and the optic nerves were collected. A : An image from the periphery of the flatmounted retinas of transduced mouse eyes that were immunostained with Brn3a immunoglobulin G (IgG) and imaged with epifluorescence microscopy. B : The morphology of the myelinated axons (toluidine blue stained) of mice 36 days after intracameral transduction with BMP2. The data are representative of images taken of four mice.

    Techniques Used: Epifluorescence Microscopy, Staining, Mouse Assay, Transduction

    2) Product Images from "The FGFR/MEK/ERK/brachyury pathway is critical for chordoma cell growth and survival"

    Article Title: The FGFR/MEK/ERK/brachyury pathway is critical for chordoma cell growth and survival

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgu014

    Expressions of FGF2 and FGFR/MEK/ERK/brachyury in chordoma cells. ( A ) Protein extracts from JHC7, UCH1 and UCH2 cells were used for western blot analysis of FGFR1, 2, 3 and 4, MEK phosphorylation (p-MEK 1/2), MEK 1/2, ERK phosphorylation (p-ERK 1/2), ERK (ERK 1/2) and brachyury. ( B ) Human foreskin fibroblast BJ cells (as a control) and chordoma cells (JHC7, UCH1 and UCH2) were cultured for 3 days in serum-free medium. FGF2 secreted into the medium was measured by ELISA and normalized to protein measured in cell lysates. ( C ) JHC7, UCH1 and UCH2 cells was incubated in serum-free medium (control) and treated with neutralizing antibody to FGF2 (FGF2 antibody, 20 µg/ml) or normal IgG (20 µg/ml) for 48h. Protein extracts from cells were used for western blot analysis of FGF2, MEK phosphorylation (p-MEK 1/2), MEK 1/2, ERK phosphorylation (p-ERK 1/2), ERK (ERK 1/2) and brachyury. ( D , E and F ) JHC7, UCH1 and UCH2 cells were incubated in triplicate serum-free medium (control) and treated with neutralizing antibody to FGF2 (FGF2 antibody, 20 µg/ml) or normal IgG (20 µg/ml) as negative control for 48h (D E) or 5 days (F). Caspase 3 activity (D), DNA fragmentation (E) and cell growth (F) were measured by the Caspase-GloR 3/7 assay, a DNA Cell Death Detection ELISA PLUS Kit and MTS assay. Values represent means ± SD ( n = 3). * P
    Figure Legend Snippet: Expressions of FGF2 and FGFR/MEK/ERK/brachyury in chordoma cells. ( A ) Protein extracts from JHC7, UCH1 and UCH2 cells were used for western blot analysis of FGFR1, 2, 3 and 4, MEK phosphorylation (p-MEK 1/2), MEK 1/2, ERK phosphorylation (p-ERK 1/2), ERK (ERK 1/2) and brachyury. ( B ) Human foreskin fibroblast BJ cells (as a control) and chordoma cells (JHC7, UCH1 and UCH2) were cultured for 3 days in serum-free medium. FGF2 secreted into the medium was measured by ELISA and normalized to protein measured in cell lysates. ( C ) JHC7, UCH1 and UCH2 cells was incubated in serum-free medium (control) and treated with neutralizing antibody to FGF2 (FGF2 antibody, 20 µg/ml) or normal IgG (20 µg/ml) for 48h. Protein extracts from cells were used for western blot analysis of FGF2, MEK phosphorylation (p-MEK 1/2), MEK 1/2, ERK phosphorylation (p-ERK 1/2), ERK (ERK 1/2) and brachyury. ( D , E and F ) JHC7, UCH1 and UCH2 cells were incubated in triplicate serum-free medium (control) and treated with neutralizing antibody to FGF2 (FGF2 antibody, 20 µg/ml) or normal IgG (20 µg/ml) as negative control for 48h (D E) or 5 days (F). Caspase 3 activity (D), DNA fragmentation (E) and cell growth (F) were measured by the Caspase-GloR 3/7 assay, a DNA Cell Death Detection ELISA PLUS Kit and MTS assay. Values represent means ± SD ( n = 3). * P

    Techniques Used: Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, Activity Assay, MTS Assay

    Related Articles

    other:

    Article Title: Transferrin Receptor Is a Specific Ferroptosis Marker
    Article Snippet: Sections were washed with PBT for three times before incubating with goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific Cat# A-11032, RRID:AB_2534091, 1:1000 dilution) or goat anti-Rrabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217, 1:1000 dilution) at room temperature for 1 h. Slides were then washed twice with PBT three times.

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  • 92
    Thermo Fisher goat anti mouse igg1
    Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with <t>IgG</t> binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p
    Goat Anti Mouse Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher goat anti mouse igg2b cross adsorbed secondary antibody
    Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit <t>IgG</t> IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.
    Goat Anti Mouse Igg2b Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher goat anti rabbit igg alexa 488 secondary antibody
    Confirmation of the formation of squid protein-based architectures in human cells. a  (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images.  b  (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset.  c  Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images.  d  (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.
    Goat Anti Rabbit Igg Alexa 488 Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher antibody alexa fluor 594 labeled goat anti rabbit igg antibody
    Intracellular localization of TagB, TagC, and Sha determined by confocal microscopy. ( A ) Z-stack imaging showing the localization of TagB, TagC, and Sha and their respective serine active site mutant variants during interaction with 5637 bladder epithelial cells after 5 h of incubation. SPATEs were detected by <t>Alexa</t> Fluor 594 (white arrowheads, red fluorescence) using anti-mouse secondary antibody and actin was stained by Alexa Fluor 488- phalloidin (green fluorescence). Images are displayed in a 3D section view with large Z-sections in the X-Y direction (R), Z-projection in the X–Z direction (P), and Z-projection in the Y–Z direction (Q). The green and red lines in R indicate the orthogonal planes of the X–Z and Y–Z projection. For each selected section, the signal was gathered from a span of 5 μm. Scale bar: 10 µm ( B ) Quantitative analysis of fluorescence intensity of F-actin in the cells treated with native or mutant SPATEs. Analysis of fluorescence intensity was done in green channel by measuring the mean gray value on ImageJ. Data represent the mean ± SEM of at least three independent experiments. Significant differences between fluorescence intensity of each native and mutant SPATE treated cell was determined using Student’s t-test with ** p
    Antibody Alexa Fluor 594 Labeled Goat Anti Rabbit Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody alexa fluor 594 labeled goat anti rabbit igg antibody/product/Thermo Fisher
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    Image Search Results


    Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p

    Journal: Nature Communications

    Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression

    doi: 10.1038/s41467-019-09263-1

    Figure Lengend Snippet: Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p

    Article Snippet: After de-paraffinization and antigen retrieval, sections were blocked in 5% goat serum and incubated overnight with mouse monoclonal anti-CD8 (1:100, IgG1, C8/144B, #108M-98, Cell Marque) and mouse monoclonal anti-Chop (1:100, IgG2b, 9C8, #ab11419, Abcam) or mouse monoclonal anti-CD8 and rabbit polyclonal anti-CHOP/GADD153 (1:500, R-20, #sc-793, Santa Cruz Biotechnologies), followed by washes in PBS and incubation in secondary goat anti-mouse IgG1 and IgG2b or goat anti-mouse IgG1 and anti-rabbit IgG labeled with Alexa Fluor® 488 and 647 (all 1:200, ThermoFisher Scientific), respectively.

    Techniques: Expressing, Quantitation Assay, Activation Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Fluorescence, FACS, Transduction

    Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    doi: 10.15252/embj.2019102539

    Figure Lengend Snippet: Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.

    Article Snippet: These were Alexa Fluor anti‐rabbit 488 (A11034), anti‐mouse 488 (A11029), anti‐mouse IgG1 488 (A21121), anti‐mouse 568 (A11031), anti‐rabbit 568 (A11036), anti‐mouse IgG2b 568 (A21144), anti‐mouse IgG2b 594 (A21145), anti‐rabbit 633 (A21070) and anti‐mouse IgG1 647 (A21240).

    Techniques: Expressing, SDS Page, Cell Culture, Negative Control, Lysis, Western Blot, Mutagenesis

    Confirmation of the formation of squid protein-based architectures in human cells. a  (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images.  b  (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset.  c  Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images.  d  (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.

    Journal: Nature Communications

    Article Title: Cephalopod-inspired optical engineering of human cells

    doi: 10.1038/s41467-020-16151-6

    Figure Lengend Snippet: Confirmation of the formation of squid protein-based architectures in human cells. a (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images. b (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. c Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images. d (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.

    Article Snippet: The cells were thoroughly washed with PBS and incubated with a goat anti-rabbit IgG Alexa 488 secondary antibody (ThermoScientific, 11008) solution (prepared at a ratio of 1:250 in PBS containing 1% BSA) and with the nuclear stain 4′,6′-diamidino-2-phenylindole (DAPI) (ThermoScientific).

    Techniques: Fluorescence, Microscopy, Expressing, Staining, Labeling, Sequencing, Transmission Electron Microscopy

    Intracellular localization of TagB, TagC, and Sha determined by confocal microscopy. ( A ) Z-stack imaging showing the localization of TagB, TagC, and Sha and their respective serine active site mutant variants during interaction with 5637 bladder epithelial cells after 5 h of incubation. SPATEs were detected by Alexa Fluor 594 (white arrowheads, red fluorescence) using anti-mouse secondary antibody and actin was stained by Alexa Fluor 488- phalloidin (green fluorescence). Images are displayed in a 3D section view with large Z-sections in the X-Y direction (R), Z-projection in the X–Z direction (P), and Z-projection in the Y–Z direction (Q). The green and red lines in R indicate the orthogonal planes of the X–Z and Y–Z projection. For each selected section, the signal was gathered from a span of 5 μm. Scale bar: 10 µm ( B ) Quantitative analysis of fluorescence intensity of F-actin in the cells treated with native or mutant SPATEs. Analysis of fluorescence intensity was done in green channel by measuring the mean gray value on ImageJ. Data represent the mean ± SEM of at least three independent experiments. Significant differences between fluorescence intensity of each native and mutant SPATE treated cell was determined using Student’s t-test with ** p

    Journal: International Journal of Molecular Sciences

    Article Title: The Serine Protease Autotransporters TagB, TagC, and Sha from Extraintestinal Pathogenic Escherichia coli Are Internalized by Human Bladder Epithelial Cells and Cause Actin Cytoskeletal Disruption

    doi: 10.3390/ijms21093047

    Figure Lengend Snippet: Intracellular localization of TagB, TagC, and Sha determined by confocal microscopy. ( A ) Z-stack imaging showing the localization of TagB, TagC, and Sha and their respective serine active site mutant variants during interaction with 5637 bladder epithelial cells after 5 h of incubation. SPATEs were detected by Alexa Fluor 594 (white arrowheads, red fluorescence) using anti-mouse secondary antibody and actin was stained by Alexa Fluor 488- phalloidin (green fluorescence). Images are displayed in a 3D section view with large Z-sections in the X-Y direction (R), Z-projection in the X–Z direction (P), and Z-projection in the Y–Z direction (Q). The green and red lines in R indicate the orthogonal planes of the X–Z and Y–Z projection. For each selected section, the signal was gathered from a span of 5 μm. Scale bar: 10 µm ( B ) Quantitative analysis of fluorescence intensity of F-actin in the cells treated with native or mutant SPATEs. Analysis of fluorescence intensity was done in green channel by measuring the mean gray value on ImageJ. Data represent the mean ± SEM of at least three independent experiments. Significant differences between fluorescence intensity of each native and mutant SPATE treated cell was determined using Student’s t-test with ** p

    Article Snippet: This was followed by incubation with secondary antibody Alexa Fluor 594-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Confocal Microscopy, Imaging, Mutagenesis, Incubation, Fluorescence, Staining