anti goat igg ab  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Goat IgG F ab 2 Isotype Control
    Description:
    Goat IgG F ab 2 Isotype Control for Flow Ctrl
    Catalog Number:
    11301c
    Price:
    None
    Applications:
    Cell Analysis|Flow Cytometry Antibodies & Secondary Detection|Flow Cytometry
    Category:
    Antibodies Secondary Detection Reagents
    Buy from Supplier


    Structured Review

    Thermo Fisher anti goat igg ab
    Goat IgG F ab 2 Isotype Control for Flow Ctrl
    https://www.bioz.com/result/anti goat igg ab/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti goat igg ab - by Bioz Stars, 2020-09
    99/100 stars

    Images

    Related Articles

    Flow Cytometry:

    Article Title: Ets Family Protein, Erg Expression in Developing and Adult Mouse Tissues by a Highly Specific Monoclonal Antibody
    Article Snippet: .. One aliquot was stained with an IgG1 isotype control antibody (Invitrogen) and the second aliquot was stained with the anti-ERG antibody at a 1:20 dilution for 1 hour at 4o C. Then cells were washed and stained with FITC-conjugated rat anti-mouse IgG1 antibody (Clone A85-1, BD Pharmingen) for an additional 30 minutes at 4o C. Cells were then washed and analyzed by using a BD FACS Canto II flow cytometer. .. Data was collected on the total cell population and the analysis was performed by using FACS Diva software (version 5 .03) (Becton Dickinson).

    In Vitro:

    Article Title: Dual Role of the Tyrosine Kinase Syk in Regulation of Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells
    Article Snippet: .. For in vitro pDC stimulation assays CpG-A (ODN 2216), CpG-B (ODN 2006), resiquimod (R848), PMA (all InvivoGen, San Diego, USA), BDCA-2 mAb (Miltenyi Biotech, Paris, France), ILT7 (CD85g) mAb and IgG1 isotype control antibody (eBioscience) were used. .. In vitro pDC stimulation To determine cytokine production, purified primary human pDCs (in the presence of IL-3) or GEN2.2 cells were kept at a concentration of 106 cells/mL aliquoted in 100-μL quantities in 96-well round-bottom culture plates, and stimulated with 4 μg/mL CpG-A, 0.5 μg/mL CpG-B, 0.5 μM R848, 25 ng/mL PMA, 10 μg/mL of BDCA-2 or ILT7 antibody, or with HCV, or HBV overnight.

    Magnetic Beads:

    Article Title: ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor
    Article Snippet: .. Immunoprecipitation assays 2–5 ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer’s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). .. After coupling, 1 mg of the antibody/bead mixture was incubated with 1–5 mg of protein lysate overnight under rotation at 4°C.

    Cytometry:

    Article Title: Ets Family Protein, Erg Expression in Developing and Adult Mouse Tissues by a Highly Specific Monoclonal Antibody
    Article Snippet: .. One aliquot was stained with an IgG1 isotype control antibody (Invitrogen) and the second aliquot was stained with the anti-ERG antibody at a 1:20 dilution for 1 hour at 4o C. Then cells were washed and stained with FITC-conjugated rat anti-mouse IgG1 antibody (Clone A85-1, BD Pharmingen) for an additional 30 minutes at 4o C. Cells were then washed and analyzed by using a BD FACS Canto II flow cytometer. .. Data was collected on the total cell population and the analysis was performed by using FACS Diva software (version 5 .03) (Becton Dickinson).

    Immunoprecipitation:

    Article Title: ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor
    Article Snippet: .. Immunoprecipitation assays 2–5 ug of AR, ERG or IgG control antibody was coupled to 1mg of magnetic beads according to manufacturer’s protocol (Invitrogen Dynabeads Antibody Coupling kit#143.11D). .. After coupling, 1 mg of the antibody/bead mixture was incubated with 1–5 mg of protein lysate overnight under rotation at 4°C.

    Incubation:

    Article Title: MIRK/DYRK1B MEDIATES SURVIVAL DURING THE DIFFERENTIATION OF C2C12 MYOBLASTS 1
    Article Snippet: .. After 3 x 5 minute washes, cells were incubated for 30 minutes with a 1:1000 dilution of goat anti-IgG antibody Alexa Fluor conjugate (Molecular Probes). .. In some experiments, sections were incubated for 20 minutes with 2 units of Alexa Fluor 594 phalloidin (Molecular Probes) diluted in 3%BSA/PBS, and washed twice.

    Article Title: CDNF rescues motor neurons in three animal models of ALS by targeting ER stress
    Article Snippet: .. The following day, the sections were rinsed with TBS-T and incubated with Alexa 488-conjugated anti-goat IgG (1:500, Thermo fisher scientific) and Alexa 589 conjugated anti-rabbit IgG (1:500, Thermo fisher scientific). .. Fluorescence images were acquired using confocal microscopy (LSM700, Carl Zeiss).

    Article Title: Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria
    Article Snippet: .. Controls were incubated with an isotype control immunoglobulin G1 (IgG1) and IgG2a antibody (eBioscience). .. A 25% (w/v) biotin solution (Vector Laboratories) was included in the primary antibody solution, to reduce non-specific background staining.

    Activity Assay:

    Article Title: Analyses of the Pathways Involved in Early- and Late-Phase Induction of IFN-Beta during C. muridarum Infection of Oviduct Epithelial Cells
    Article Snippet: .. To neutralize IFN-β activity, cell supernatants were supplemented with either 0.1 or 1 μg/ml of rabbit anti-IFN-β polyclonal antibody (Thermo Scientific, Rockford, IL), or the corresponding IgG control antibody (Thermo Scientific, Rockford, IL). .. In the experiments to ascertain the impact of neutralizing IFN-β function at specific time-points on the total amounts of Chlamydia -induced IFN-β at 24 hours post-infection, either anti-IFN-β or IgG control antibody was added to the supernatants of infected cells at the specified time-point, followed by gentle swirling of the 24-well plate before returning to the 37°C tissue-culture incubator for the remainder of the 24h infection.

    Staining:

    Article Title: Ets Family Protein, Erg Expression in Developing and Adult Mouse Tissues by a Highly Specific Monoclonal Antibody
    Article Snippet: .. One aliquot was stained with an IgG1 isotype control antibody (Invitrogen) and the second aliquot was stained with the anti-ERG antibody at a 1:20 dilution for 1 hour at 4o C. Then cells were washed and stained with FITC-conjugated rat anti-mouse IgG1 antibody (Clone A85-1, BD Pharmingen) for an additional 30 minutes at 4o C. Cells were then washed and analyzed by using a BD FACS Canto II flow cytometer. .. Data was collected on the total cell population and the analysis was performed by using FACS Diva software (version 5 .03) (Becton Dickinson).

    FACS:

    Article Title: Ets Family Protein, Erg Expression in Developing and Adult Mouse Tissues by a Highly Specific Monoclonal Antibody
    Article Snippet: .. One aliquot was stained with an IgG1 isotype control antibody (Invitrogen) and the second aliquot was stained with the anti-ERG antibody at a 1:20 dilution for 1 hour at 4o C. Then cells were washed and stained with FITC-conjugated rat anti-mouse IgG1 antibody (Clone A85-1, BD Pharmingen) for an additional 30 minutes at 4o C. Cells were then washed and analyzed by using a BD FACS Canto II flow cytometer. .. Data was collected on the total cell population and the analysis was performed by using FACS Diva software (version 5 .03) (Becton Dickinson).

    Injection:

    Article Title: Clinical and Experimental Pancreatic Islet Transplantation to Striated Muscle
    Article Snippet: .. Immunoglobulin G (IgG) isotype antibody (eBioscience) was injected in controls. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher alexa fluor 488 goat anti mouse igg antibody
    STIM2 recruits CaMKK2 to regulate calcium-induced AMPK activation. A ) STIM2 interacts with CaMKK2. HEK 293T cells were cotransfected with STIM2-SFB [or empty vector (EV)] and myc-CaMKK2. STIM2-SFB was immunoprecipitated using S-protein beads, and coprecipitated myc-CaMKK2 was detected after immunoblotting using anti-myc antibody. B , C ) Cytosolic calcium influx promotes STIM2 interaction with CaMKK2. HEK 293T ( B ) and HeLa ( C ) cells stably expressing STIM2-SFB alone were transiently transfected with myc-CaMKK2 and then incubated with medium containing thapsigargin (1 μM) for 30 min. Cell lysates were subjected to immunoprecipitation using S-protein beads. Coprecipitates were analyzed after Western blotting with anti-myc antibody. D ) STIM2 interacts with CaMKK2 through its CAD/SOAR region. Upper panel: Schematic representation of STIM2 full-length and deletion mutants used in this study. Lower panel: Protein extracts of HEK 293T cells expressing full-length and truncated mutants of STIM2-SFB along with myc-CaMKK2 were immunoprecipitated with S-protein beads and analyzed for coprecipitated myc-CaMKK2 using immunoblotting with anti-myc antibody. E ) AMPK does not form a stable complex with CaMKK2. Protein extracts of HEK 293T cells expressing full-length CaMKK2-SFB were immunoprecipitated with S-protein beads and analyzed for coprecipitated proteins using indicated antibodies. F ) Calcium-induced colocalization between AMPK and CaMKK2 depends on STIM2. Control and STIM2-depleted HeLa cells were transiently transfected with myc-CaMKK2, incubated with medium containing thapsigargin (1 μM) for 30 min, and processed for immunofluorescence imaging. Rabbit anti-myc antibody, goat anti-rabbit <t>IgG</t> <t>Alexa</t> Fluor 633 secondary antibody for myc (red), and mouse anti–AMPK-α goat anti-mouse IgG Alexa <t>Fluor</t> 488 secondary for AMPK-α (green) were used. Representative images from each condition are presented. Overlap coefficients for the AMPK and CaMKK2 signal are presented in the upper right corner of the merge panels ( n = 19 for Ctl sg + DMSO, n = 22 for Ctl sg + thapsigargin, and n = 18 for STIM2 sg + thapsigargin). In all images, insets display the selected area along with the overlays. Scale bars, 5 μm. P
    Alexa Fluor 488 Goat Anti Mouse Igg Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 goat anti mouse igg antibody/product/Thermo Fisher
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 goat anti mouse igg antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    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 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igg1/product/Thermo Fisher
    Average 92 stars, based on 99 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igg1 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/goat anti mouse igg2b cross adsorbed secondary antibody/product/Thermo Fisher
    Average 89 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igg2b cross adsorbed secondary antibody - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    Image Search Results


    STIM2 recruits CaMKK2 to regulate calcium-induced AMPK activation. A ) STIM2 interacts with CaMKK2. HEK 293T cells were cotransfected with STIM2-SFB [or empty vector (EV)] and myc-CaMKK2. STIM2-SFB was immunoprecipitated using S-protein beads, and coprecipitated myc-CaMKK2 was detected after immunoblotting using anti-myc antibody. B , C ) Cytosolic calcium influx promotes STIM2 interaction with CaMKK2. HEK 293T ( B ) and HeLa ( C ) cells stably expressing STIM2-SFB alone were transiently transfected with myc-CaMKK2 and then incubated with medium containing thapsigargin (1 μM) for 30 min. Cell lysates were subjected to immunoprecipitation using S-protein beads. Coprecipitates were analyzed after Western blotting with anti-myc antibody. D ) STIM2 interacts with CaMKK2 through its CAD/SOAR region. Upper panel: Schematic representation of STIM2 full-length and deletion mutants used in this study. Lower panel: Protein extracts of HEK 293T cells expressing full-length and truncated mutants of STIM2-SFB along with myc-CaMKK2 were immunoprecipitated with S-protein beads and analyzed for coprecipitated myc-CaMKK2 using immunoblotting with anti-myc antibody. E ) AMPK does not form a stable complex with CaMKK2. Protein extracts of HEK 293T cells expressing full-length CaMKK2-SFB were immunoprecipitated with S-protein beads and analyzed for coprecipitated proteins using indicated antibodies. F ) Calcium-induced colocalization between AMPK and CaMKK2 depends on STIM2. Control and STIM2-depleted HeLa cells were transiently transfected with myc-CaMKK2, incubated with medium containing thapsigargin (1 μM) for 30 min, and processed for immunofluorescence imaging. Rabbit anti-myc antibody, goat anti-rabbit IgG Alexa Fluor 633 secondary antibody for myc (red), and mouse anti–AMPK-α goat anti-mouse IgG Alexa Fluor 488 secondary for AMPK-α (green) were used. Representative images from each condition are presented. Overlap coefficients for the AMPK and CaMKK2 signal are presented in the upper right corner of the merge panels ( n = 19 for Ctl sg + DMSO, n = 22 for Ctl sg + thapsigargin, and n = 18 for STIM2 sg + thapsigargin). In all images, insets display the selected area along with the overlays. Scale bars, 5 μm. P

    Journal: The FASEB Journal

    Article Title: STIM2 interacts with AMPK and regulates calcium-induced AMPK activation

    doi: 10.1096/fj.201801225R

    Figure Lengend Snippet: STIM2 recruits CaMKK2 to regulate calcium-induced AMPK activation. A ) STIM2 interacts with CaMKK2. HEK 293T cells were cotransfected with STIM2-SFB [or empty vector (EV)] and myc-CaMKK2. STIM2-SFB was immunoprecipitated using S-protein beads, and coprecipitated myc-CaMKK2 was detected after immunoblotting using anti-myc antibody. B , C ) Cytosolic calcium influx promotes STIM2 interaction with CaMKK2. HEK 293T ( B ) and HeLa ( C ) cells stably expressing STIM2-SFB alone were transiently transfected with myc-CaMKK2 and then incubated with medium containing thapsigargin (1 μM) for 30 min. Cell lysates were subjected to immunoprecipitation using S-protein beads. Coprecipitates were analyzed after Western blotting with anti-myc antibody. D ) STIM2 interacts with CaMKK2 through its CAD/SOAR region. Upper panel: Schematic representation of STIM2 full-length and deletion mutants used in this study. Lower panel: Protein extracts of HEK 293T cells expressing full-length and truncated mutants of STIM2-SFB along with myc-CaMKK2 were immunoprecipitated with S-protein beads and analyzed for coprecipitated myc-CaMKK2 using immunoblotting with anti-myc antibody. E ) AMPK does not form a stable complex with CaMKK2. Protein extracts of HEK 293T cells expressing full-length CaMKK2-SFB were immunoprecipitated with S-protein beads and analyzed for coprecipitated proteins using indicated antibodies. F ) Calcium-induced colocalization between AMPK and CaMKK2 depends on STIM2. Control and STIM2-depleted HeLa cells were transiently transfected with myc-CaMKK2, incubated with medium containing thapsigargin (1 μM) for 30 min, and processed for immunofluorescence imaging. Rabbit anti-myc antibody, goat anti-rabbit IgG Alexa Fluor 633 secondary antibody for myc (red), and mouse anti–AMPK-α goat anti-mouse IgG Alexa Fluor 488 secondary for AMPK-α (green) were used. Representative images from each condition are presented. Overlap coefficients for the AMPK and CaMKK2 signal are presented in the upper right corner of the merge panels ( n = 19 for Ctl sg + DMSO, n = 22 for Ctl sg + thapsigargin, and n = 18 for STIM2 sg + thapsigargin). In all images, insets display the selected area along with the overlays. Scale bars, 5 μm. P

    Article Snippet: After incubation with primary antibodies, cells were washed 3 times and incubated with the following secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG antibody (A-11001, 1:500 dilution; Thermo Fisher Scientific) and Alexa Fluor 633 goat anti-rabbit IgG antibody (A21070; Thermo Fisher Scientific).

    Techniques: Activation Assay, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing, Transfection, Incubation, Western Blot, Immunofluorescence, Imaging, CTL Assay

    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

    Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intramuscular (IM) immunization. Groups of 10 female BALB/c mice were immunized with PBS or 10 10 vp of the indicated SC-Ads. Six weeks later, samples were collected for ELISA vs. clade C CN54 gp140 ( A ) , ( B ), and ( C ) six week ELISAs after a single high dose IM immunization. Mean +/− SEM is shown. ( A ) 1/2000 sera dilutions detecting IgG. ( B ) IgG ELISA for 1/35 dilution of vaginal wash samples. ( C ) IgA ELISA for 1/35 dilution of vaginal wash samples. * p

    Journal: Vaccines

    Article Title: Genetic Adjuvants in Replicating Single-Cycle Adenovirus Vectors Amplify Systemic and Mucosal Immune Responses against HIV-1 Envelope

    doi: 10.3390/vaccines8010064

    Figure Lengend Snippet: Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intramuscular (IM) immunization. Groups of 10 female BALB/c mice were immunized with PBS or 10 10 vp of the indicated SC-Ads. Six weeks later, samples were collected for ELISA vs. clade C CN54 gp140 ( A ) , ( B ), and ( C ) six week ELISAs after a single high dose IM immunization. Mean +/− SEM is shown. ( A ) 1/2000 sera dilutions detecting IgG. ( B ) IgG ELISA for 1/35 dilution of vaginal wash samples. ( C ) IgA ELISA for 1/35 dilution of vaginal wash samples. * p

    Article Snippet: The wells were washed, and goat anti-mouse IgG or IgA horseradish peroxidase (Thermo Fisher Scientific Inc.) was added and incubated 2 h at RT.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intranasal (IN) immunization. Groups of 10 female BALB/c mice were immunized with phosphate-buffered saline (PBS) or 10 9 vp of the indicated SC-Ads. Six weeks later, samples were collected for enzyme-linked immunosorbent assay (ELISA) vs. clade C CN54 gp140. ( A ) Sub-isotyping ELISA for the indicated samples at 1/200 dilution (low dilution used for low-sensitivity sub-isotyping kit). All IgG isotypes in the granulocyte macrophage colony-stimulating factor (GMCSF) and TcdA/B groups were significantly different than PBS by 2-way ANOVA. ( B ) ELISA OD450 levels are shown for 1/35 dilution of vaginal wash samples with detection by anti-IgA. * p

    Journal: Vaccines

    Article Title: Genetic Adjuvants in Replicating Single-Cycle Adenovirus Vectors Amplify Systemic and Mucosal Immune Responses against HIV-1 Envelope

    doi: 10.3390/vaccines8010064

    Figure Lengend Snippet: Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intranasal (IN) immunization. Groups of 10 female BALB/c mice were immunized with phosphate-buffered saline (PBS) or 10 9 vp of the indicated SC-Ads. Six weeks later, samples were collected for enzyme-linked immunosorbent assay (ELISA) vs. clade C CN54 gp140. ( A ) Sub-isotyping ELISA for the indicated samples at 1/200 dilution (low dilution used for low-sensitivity sub-isotyping kit). All IgG isotypes in the granulocyte macrophage colony-stimulating factor (GMCSF) and TcdA/B groups were significantly different than PBS by 2-way ANOVA. ( B ) ELISA OD450 levels are shown for 1/35 dilution of vaginal wash samples with detection by anti-IgA. * p

    Article Snippet: The wells were washed, and goat anti-mouse IgG or IgA horseradish peroxidase (Thermo Fisher Scientific Inc.) was added and incubated 2 h at RT.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    IM or IN SOSIP protein boost of SC-Ad-env + SC-Ad-genetic adjuvants. The groups of 10 mice from Figure 3 were divided and boosted with 5 µg CZA clade C SOSIP protein adjuvanted with 1 µg alphaGalCer by either the IM or IN route. Two weeks later, 1/35 dilutions of vaginal washes were assay for anti-CN54 IgG or IgA antibodies by ELISA.

    Journal: Vaccines

    Article Title: Genetic Adjuvants in Replicating Single-Cycle Adenovirus Vectors Amplify Systemic and Mucosal Immune Responses against HIV-1 Envelope

    doi: 10.3390/vaccines8010064

    Figure Lengend Snippet: IM or IN SOSIP protein boost of SC-Ad-env + SC-Ad-genetic adjuvants. The groups of 10 mice from Figure 3 were divided and boosted with 5 µg CZA clade C SOSIP protein adjuvanted with 1 µg alphaGalCer by either the IM or IN route. Two weeks later, 1/35 dilutions of vaginal washes were assay for anti-CN54 IgG or IgA antibodies by ELISA.

    Article Snippet: The wells were washed, and goat anti-mouse IgG or IgA horseradish peroxidase (Thermo Fisher Scientific Inc.) was added and incubated 2 h at RT.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    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