Structured Review

Enzo Biochem brefeldin a
ExoU PLA 2 activity stimulates IL-8 secretion by P. aeruginosa -infected A549 cultures. In (A) and (B), cells were infected with the different P. aeruginosa strains for 21 hours and the concentrations of IL-8 in supernatants were assessed by ELISA. In (B), <t>Brefeldin</t> A was added or not to the gentamicin-containing culture medium. The graphs show the means ± SEM of three assays performed in quadruplicate. ***p
Brefeldin A, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 97/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "ExoU Activates NF-?B and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection"

Article Title: ExoU Activates NF-?B and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041772

ExoU PLA 2 activity stimulates IL-8 secretion by P. aeruginosa -infected A549 cultures. In (A) and (B), cells were infected with the different P. aeruginosa strains for 21 hours and the concentrations of IL-8 in supernatants were assessed by ELISA. In (B), Brefeldin A was added or not to the gentamicin-containing culture medium. The graphs show the means ± SEM of three assays performed in quadruplicate. ***p
Figure Legend Snippet: ExoU PLA 2 activity stimulates IL-8 secretion by P. aeruginosa -infected A549 cultures. In (A) and (B), cells were infected with the different P. aeruginosa strains for 21 hours and the concentrations of IL-8 in supernatants were assessed by ELISA. In (B), Brefeldin A was added or not to the gentamicin-containing culture medium. The graphs show the means ± SEM of three assays performed in quadruplicate. ***p

Techniques Used: Proximity Ligation Assay, Activity Assay, Infection, Enzyme-linked Immunosorbent Assay

2) Product Images from "Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus"

Article Title: Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

Journal: mBio

doi: 10.1128/mBio.01060-20

SrcA, PmrA, and the canonical UPR protect against calcineurin inhibition. (A) Serial 10-fold dilutions of conidia from the KU70 parental strain and the Δ hacA mutant were incubated for 2 days at 37°C on AMM plates without or with supplementation with Ca 2+ and in the presence or absence of brefeldin A (BFA), calcofluor white (CFW), or caspofungin (CAS). (B) The indicated strains were incubated for 2 days at 37°C on AMM plates in the presence of cyclosporine (CsA) or FK506. (C) Bright-field and PI-stained fluorescent images of hyphae after 24 h at 37°C in liquid AMM containing CsA or FK506. No fluorescence was observed in untreated controls (see Fig. 8B ). Bars: 50 μm. (D) Serial 10-fold dilutions of conidia from mutants lacking srcA and/or pmrA and the KU80 parental strain were spotted onto AMM in the presence or absence of FK506 or CsA and incubated for 2 days at 37°C.
Figure Legend Snippet: SrcA, PmrA, and the canonical UPR protect against calcineurin inhibition. (A) Serial 10-fold dilutions of conidia from the KU70 parental strain and the Δ hacA mutant were incubated for 2 days at 37°C on AMM plates without or with supplementation with Ca 2+ and in the presence or absence of brefeldin A (BFA), calcofluor white (CFW), or caspofungin (CAS). (B) The indicated strains were incubated for 2 days at 37°C on AMM plates in the presence of cyclosporine (CsA) or FK506. (C) Bright-field and PI-stained fluorescent images of hyphae after 24 h at 37°C in liquid AMM containing CsA or FK506. No fluorescence was observed in untreated controls (see Fig. 8B ). Bars: 50 μm. (D) Serial 10-fold dilutions of conidia from mutants lacking srcA and/or pmrA and the KU80 parental strain were spotted onto AMM in the presence or absence of FK506 or CsA and incubated for 2 days at 37°C.

Techniques Used: Subrenal Capsule Assay, Inhibition, Mutagenesis, Incubation, Staining, Fluorescence

3) Product Images from "Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation"

Article Title: Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation

Journal: Nature communications

doi: 10.1038/ncomms4551

T cells require Bhlhe40 for normal cytokine production after immunization a , b , ELISPOT assays for the quantitation of cells secreting ( a ) IL-2, IFN-γ, and IL-17A or ( b ) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40 −/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c , e , DLN cells from immunized WT and Bhlhe40 −/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. ( c ) GM-CSF or ( e ) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. f , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. g , Frequencies of GM-CSF + and IL-17A + γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40 −/− mice (n=3 per group) as determined by ICS. h , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.
Figure Legend Snippet: T cells require Bhlhe40 for normal cytokine production after immunization a , b , ELISPOT assays for the quantitation of cells secreting ( a ) IL-2, IFN-γ, and IL-17A or ( b ) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40 −/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c , e , DLN cells from immunized WT and Bhlhe40 −/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. ( c ) GM-CSF or ( e ) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. f , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. g , Frequencies of GM-CSF + and IL-17A + γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40 −/− mice (n=3 per group) as determined by ICS. h , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.

Techniques Used: Enzyme-linked Immunospot, Quantitation Assay, Mouse Assay, Cell Culture, Staining

4) Product Images from "Functional characterization of the lysosomal membrane protein TMEM192 in mice"

Article Title: Functional characterization of the lysosomal membrane protein TMEM192 in mice

Journal: Oncotarget

doi: 10.18632/oncotarget.17514

Proteolytic processing of murine TMEM192 occurs in lysosomes A . Abundance of the TMEM192 protein (closed arrow-head) and the derived TMEM192 N-terminal fragment (NTF, open arrow-head) was compared in murine embryonic fibroblasts (MEF), RAW 264.7 cells, N2A cells and primary Bone-marrow-derived macrophages from wild type mice ( +/+ ). As a control for antibody specificity, macrophages from TMEM192 -/- were included in the analysis. Total cell lysates of the depicted cell lines were subjected to Western blotting using the TMEM192 antibody. Equal loading was confirmed by re-probing the membrane with anti-GAPDH antibody. B . HeLa cells were transiently transfected with murine TMEM192. Cells were treated overnight with Bafilomycin (30 nM), NH 4 Cl (25 mM), Leupeptin (100 µM), E64d (40 µM), Brefeldin A (1 µg/ml) or H 2 O or DMSO as negative control. Aliquots of total cell lysates were analysed by Western blotting using the TMEM192 antibody. Due to a low processing efficiency of TMEM192 upon overexpression conditions, a longer exposure of the membrane was included to visualize the NTF and the effect of the different inhibitors on the generation of this fragment.
Figure Legend Snippet: Proteolytic processing of murine TMEM192 occurs in lysosomes A . Abundance of the TMEM192 protein (closed arrow-head) and the derived TMEM192 N-terminal fragment (NTF, open arrow-head) was compared in murine embryonic fibroblasts (MEF), RAW 264.7 cells, N2A cells and primary Bone-marrow-derived macrophages from wild type mice ( +/+ ). As a control for antibody specificity, macrophages from TMEM192 -/- were included in the analysis. Total cell lysates of the depicted cell lines were subjected to Western blotting using the TMEM192 antibody. Equal loading was confirmed by re-probing the membrane with anti-GAPDH antibody. B . HeLa cells were transiently transfected with murine TMEM192. Cells were treated overnight with Bafilomycin (30 nM), NH 4 Cl (25 mM), Leupeptin (100 µM), E64d (40 µM), Brefeldin A (1 µg/ml) or H 2 O or DMSO as negative control. Aliquots of total cell lysates were analysed by Western blotting using the TMEM192 antibody. Due to a low processing efficiency of TMEM192 upon overexpression conditions, a longer exposure of the membrane was included to visualize the NTF and the effect of the different inhibitors on the generation of this fragment.

Techniques Used: Derivative Assay, Mouse Assay, Western Blot, Transfection, Negative Control, Over Expression

5) Product Images from "Rho and Rac, but not ROCK, are required for secretion of human and mouse eosinophil-associated RNases"

Article Title: Rho and Rac, but not ROCK, are required for secretion of human and mouse eosinophil-associated RNases

Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

doi: 10.1111/cea.13292

CCL11-mediated EAR secretion from eosinophils is nocodazole and brefeldin A- dependent (A, B) RNase activity was measured in supernatants of mCCL11- (A) or ,CCL24- (B) stimulated mouse eosinophils in the presence of the microtubule polymerization blocker, nocodazole, as described in Material and Methods. Data are mean ± SD of four independent experiments (C) RNase activity was measured in supernatants of hCCL11-stimulated human eosinophils in the presence of the microtubule polymerization blocker, nocodazole. Data are mean ± SD of three independent experiments. (D) RNase activity was measured in supernatants of mCCL11- or ,CCL24- stimulated mouse eosinophils in the presence of the microtubule polymerization blocker, brefeldin A (BFA), as described in Material and Methods. Data are mean of two independent experiments. Higher RNase activity values were obtained in D, due to higher numbers of cells (1× 10 6 cells per sample) used for the experiments. Asterisks represent P values
Figure Legend Snippet: CCL11-mediated EAR secretion from eosinophils is nocodazole and brefeldin A- dependent (A, B) RNase activity was measured in supernatants of mCCL11- (A) or ,CCL24- (B) stimulated mouse eosinophils in the presence of the microtubule polymerization blocker, nocodazole, as described in Material and Methods. Data are mean ± SD of four independent experiments (C) RNase activity was measured in supernatants of hCCL11-stimulated human eosinophils in the presence of the microtubule polymerization blocker, nocodazole. Data are mean ± SD of three independent experiments. (D) RNase activity was measured in supernatants of mCCL11- or ,CCL24- stimulated mouse eosinophils in the presence of the microtubule polymerization blocker, brefeldin A (BFA), as described in Material and Methods. Data are mean of two independent experiments. Higher RNase activity values were obtained in D, due to higher numbers of cells (1× 10 6 cells per sample) used for the experiments. Asterisks represent P values

Techniques Used: Activity Assay

6) Product Images from "IL-27p28 Production by XCR1+ Dendritic Cells and Monocytes Effectively Predicts Adjuvant-Elicited CD8+ T Cell Responses"

Article Title: IL-27p28 Production by XCR1+ Dendritic Cells and Monocytes Effectively Predicts Adjuvant-Elicited CD8+ T Cell Responses

Journal: ImmunoHorizons

doi: 10.4049/immunohorizons.1700054

The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 +  T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b  tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 +  spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C  and  D ) The number of tetramer +  T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP +  cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in  Fig. 3 .  r 2  and  p  values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in  Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/−  DCs, as in  Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 +  XCR1 +  DCs is shown. ( F ) Tetramer +  T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 +  cells shown in (E).  r 2  and  p  values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p
Figure Legend Snippet: The magnitude of IL-27 produced by cDC1 cells predicts the magnitude of the vaccine adjuvant-elicited CD8 + T cell response Mice were immunized i.v. with OVA plus the indicated adjuvants. Seven days later, the spleens were harvested and stained with SIINFEKL–MHC class I K b tetramers, as previously described ( 20 ), to identify OVA-specific T cells. ( A ) Representative dot plots showing tetramer staining on all live singlet B220 − CD8 + CD3 + spleen cells isolated from mice immunized with the indicated adjuvants. ( B ) Quantification of T cell data shown in (A) after immunization with the indicated adjuvants. Error bars indicate SD derived from three mice each. ( C and D ) The number of tetramer + T cells generated by each adjuvant, as shown in (B), was plotted against eGFP gMFI of cDC1 cells (C) or the percentage of eGFP + cDC1 cells (D) 6 h postimmunization with each adjuvant, as shown in Fig. 3 . r 2 and p values were determined by linear regression analysis (Prism). ( E ) Mice were challenged with the indicated innate stimuli, and the splenocytes were isolated at 6 h, as in Fig. 3 . Spleens were incubated in vitro for 6 h in the presence of brefeldin A, after which they were stained to identify XCR1 +/− DCs, as in Fig. 2 . The cells were then fixed, permeabilized, and stained for intracellular IL-12. The percentage of IL-12 + XCR1 + DCs is shown. ( F ) Tetramer + T cell numbers in the spleen 7 d after immunization with the indicated adjuvants were plotted against the percentage of IL-12 + cells shown in (E). r 2 and p values were determined by linear regression analysis (Prism). Data in (E) and (F) are from one of two experiments performed. ( G ) Mice were immunized with OVA + LTA, as in (A), with or without the addition of 10 μg of rIL-27. T cell responses were measured as in (A) and (B). * p

Techniques Used: Produced, Mouse Assay, Staining, Isolation, Derivative Assay, Generated, Incubation, In Vitro

7) Product Images from "Pathogen-Reactive T Helper Cell Analysis in the Pig"

Article Title: Pathogen-Reactive T Helper Cell Analysis in the Pig

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00565

Ide Ssuis -reactive Th cells are increased in PBMCs of immunized piglets identified by CD154 expression and cytokine production and the frequency of CD154 + IFN-γ + Th cells correlates with Ide Ssuis -specific IgG . (A) Piglets were primed with rIde Ssuis followed by booster immunization 2 weeks later. Blood samples for the investigation of antigen-specific Th cells were taken 2 week post-booster immunization. (B,C) To detect antigen-reactive Th cells, PBMCs derived from rIde Ssuis -immunized (imm; n = 8) or placebo-treated (plac; n = 7) piglets were ex vivo restimulated for 18 h with 5 µg/ml rIde Ssuis or ctr-antigen (rSfb I), respectively, in presence of Brefeldin A (2 µg/ml) for the last 4 h. The frequency of antigen-specific Th cells was calculated as the difference of specified cells from antigen-restimulated and medium-cultivated PBMC, respectively. Statistical analysis was performed with Kruskal–Wallis test (** p = 0.0063) and Dunn’s multiple comparison test (* p ≤ 0.05). Ide Ssuis specific-IgGs were measured by ELISA with serum from the same piglets before and after immunization, using an independent Ide Ssuis -IgG positive reference serum. The correlation between Ide Ssuis -IgG and frequency of Ide Ssuis induced CD154 + IFN-γ + Th cells (D) or frequency of ctr-ag induced CD154 + IFN-γ + (E) was estimated by Pearson correlation (* p ≤ 0.05).
Figure Legend Snippet: Ide Ssuis -reactive Th cells are increased in PBMCs of immunized piglets identified by CD154 expression and cytokine production and the frequency of CD154 + IFN-γ + Th cells correlates with Ide Ssuis -specific IgG . (A) Piglets were primed with rIde Ssuis followed by booster immunization 2 weeks later. Blood samples for the investigation of antigen-specific Th cells were taken 2 week post-booster immunization. (B,C) To detect antigen-reactive Th cells, PBMCs derived from rIde Ssuis -immunized (imm; n = 8) or placebo-treated (plac; n = 7) piglets were ex vivo restimulated for 18 h with 5 µg/ml rIde Ssuis or ctr-antigen (rSfb I), respectively, in presence of Brefeldin A (2 µg/ml) for the last 4 h. The frequency of antigen-specific Th cells was calculated as the difference of specified cells from antigen-restimulated and medium-cultivated PBMC, respectively. Statistical analysis was performed with Kruskal–Wallis test (** p = 0.0063) and Dunn’s multiple comparison test (* p ≤ 0.05). Ide Ssuis specific-IgGs were measured by ELISA with serum from the same piglets before and after immunization, using an independent Ide Ssuis -IgG positive reference serum. The correlation between Ide Ssuis -IgG and frequency of Ide Ssuis induced CD154 + IFN-γ + Th cells (D) or frequency of ctr-ag induced CD154 + IFN-γ + (E) was estimated by Pearson correlation (* p ≤ 0.05).

Techniques Used: Expressing, Derivative Assay, Ex Vivo, Enzyme-linked Immunosorbent Assay

8) Product Images from "Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge"

Article Title: Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

Journal: Developmental cell

doi: 10.1016/j.devcel.2013.08.019

Cortical ER is recruited to the WRAMP structure (A) EM tomography showing the WRAMP structure in a cell treated with Wnt5a. Colors indicate MVBs (blue) and microfilaments (yellow) interspersed among ER tubules and sheets (red) which extend toward the edge of the cell. (B) Immuno-EM using anti-GFP antibodies labeled with gold particles reveals the presence of MCAM-GFP (arrows) within intralumenal vesicles and limiting membranes of MVBs, and at the plasma membrane. Bottom panels show magnified boxes in white. (C) EM tomographs indicate cortical ER (red) juxtaposed within 10 nm of the plasma membrane (white). (D) ). (E) Indirect immunofluorescense reveals localization of endogeneous calnexin to the WRAMP structure. ( F ) Pretreatment of cells with Brefeldin A or RNAi knockdown of COP-Iβ blocks WRAMP structure formation. Cells on plates were quantified for the WRAMP structure, monitored by endogenous MCAM polarization (± s.d., n=3, each counting 200–350 cells)
Figure Legend Snippet: Cortical ER is recruited to the WRAMP structure (A) EM tomography showing the WRAMP structure in a cell treated with Wnt5a. Colors indicate MVBs (blue) and microfilaments (yellow) interspersed among ER tubules and sheets (red) which extend toward the edge of the cell. (B) Immuno-EM using anti-GFP antibodies labeled with gold particles reveals the presence of MCAM-GFP (arrows) within intralumenal vesicles and limiting membranes of MVBs, and at the plasma membrane. Bottom panels show magnified boxes in white. (C) EM tomographs indicate cortical ER (red) juxtaposed within 10 nm of the plasma membrane (white). (D) ). (E) Indirect immunofluorescense reveals localization of endogeneous calnexin to the WRAMP structure. ( F ) Pretreatment of cells with Brefeldin A or RNAi knockdown of COP-Iβ blocks WRAMP structure formation. Cells on plates were quantified for the WRAMP structure, monitored by endogenous MCAM polarization (± s.d., n=3, each counting 200–350 cells)

Techniques Used: Labeling

Related Articles

Recombinant:

Article Title: Pathogen-Reactive T Helper Cell Analysis in the Pig
Article Snippet: .. Following a resting time (2–4 h), PBMCs were stimulated for 18 h with 5 µg/ml rIdeSsuis or with control-antigen (ctr-ag) Sfb I (fibronectin-binding protein of Streptococcus pyogenes ), and in presence of Brefeldin A (2 µg/ml, Enzo Life Sciences) for the last 4 h. Recombinant IdeSsuis was expressed in E. coli and purified as described previously ( ). ..

Purification:

Article Title: Pathogen-Reactive T Helper Cell Analysis in the Pig
Article Snippet: .. Following a resting time (2–4 h), PBMCs were stimulated for 18 h with 5 µg/ml rIdeSsuis or with control-antigen (ctr-ag) Sfb I (fibronectin-binding protein of Streptococcus pyogenes ), and in presence of Brefeldin A (2 µg/ml, Enzo Life Sciences) for the last 4 h. Recombinant IdeSsuis was expressed in E. coli and purified as described previously ( ). ..

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    Enzo Biochem brefeldin a
    SrcA, PmrA, and the canonical UPR protect against calcineurin inhibition. (A) Serial 10-fold dilutions of conidia from the KU70 parental strain and the Δ hacA mutant were incubated for 2 days at 37°C on AMM plates without or with supplementation with Ca 2+ and in the presence or absence of <t>brefeldin</t> A (BFA), calcofluor white (CFW), or caspofungin (CAS). (B) The indicated strains were incubated for 2 days at 37°C on AMM plates in the presence of cyclosporine (CsA) or FK506. (C) Bright-field and PI-stained fluorescent images of hyphae after 24 h at 37°C in liquid AMM containing CsA or FK506. No fluorescence was observed in untreated controls (see Fig. 8B ). Bars: 50 μm. (D) Serial 10-fold dilutions of conidia from mutants lacking srcA and/or pmrA and the KU80 parental strain were spotted onto AMM in the presence or absence of FK506 or CsA and incubated for 2 days at 37°C.
    Brefeldin A, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 97/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a/product/Enzo Biochem
    Average 97 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
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    SrcA, PmrA, and the canonical UPR protect against calcineurin inhibition. (A) Serial 10-fold dilutions of conidia from the KU70 parental strain and the Δ hacA mutant were incubated for 2 days at 37°C on AMM plates without or with supplementation with Ca 2+ and in the presence or absence of brefeldin A (BFA), calcofluor white (CFW), or caspofungin (CAS). (B) The indicated strains were incubated for 2 days at 37°C on AMM plates in the presence of cyclosporine (CsA) or FK506. (C) Bright-field and PI-stained fluorescent images of hyphae after 24 h at 37°C in liquid AMM containing CsA or FK506. No fluorescence was observed in untreated controls (see Fig. 8B ). Bars: 50 μm. (D) Serial 10-fold dilutions of conidia from mutants lacking srcA and/or pmrA and the KU80 parental strain were spotted onto AMM in the presence or absence of FK506 or CsA and incubated for 2 days at 37°C.

    Journal: mBio

    Article Title: Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

    doi: 10.1128/mBio.01060-20

    Figure Lengend Snippet: SrcA, PmrA, and the canonical UPR protect against calcineurin inhibition. (A) Serial 10-fold dilutions of conidia from the KU70 parental strain and the Δ hacA mutant were incubated for 2 days at 37°C on AMM plates without or with supplementation with Ca 2+ and in the presence or absence of brefeldin A (BFA), calcofluor white (CFW), or caspofungin (CAS). (B) The indicated strains were incubated for 2 days at 37°C on AMM plates in the presence of cyclosporine (CsA) or FK506. (C) Bright-field and PI-stained fluorescent images of hyphae after 24 h at 37°C in liquid AMM containing CsA or FK506. No fluorescence was observed in untreated controls (see Fig. 8B ). Bars: 50 μm. (D) Serial 10-fold dilutions of conidia from mutants lacking srcA and/or pmrA and the KU80 parental strain were spotted onto AMM in the presence or absence of FK506 or CsA and incubated for 2 days at 37°C.

    Article Snippet: The chemicals used to induce stress included dithiothreitol (Thermo Scientific), tunicamycin (Cayman Chemical), brefeldin A (Enzo), calcofluor white (Sigma), Congo red (Sigma), hygromycin B (RPI), BAPTA (Invitrogen), calcimycin [A23187] (Sigma), amiodarone (Sigma), cyclosporine (InvivoGen), and FK506 (InvivoGen).

    Techniques: Subrenal Capsule Assay, Inhibition, Mutagenesis, Incubation, Staining, Fluorescence

    Proteolytic processing of murine TMEM192 occurs in lysosomes A . Abundance of the TMEM192 protein (closed arrow-head) and the derived TMEM192 N-terminal fragment (NTF, open arrow-head) was compared in murine embryonic fibroblasts (MEF), RAW 264.7 cells, N2A cells and primary Bone-marrow-derived macrophages from wild type mice ( +/+ ). As a control for antibody specificity, macrophages from TMEM192 -/- were included in the analysis. Total cell lysates of the depicted cell lines were subjected to Western blotting using the TMEM192 antibody. Equal loading was confirmed by re-probing the membrane with anti-GAPDH antibody. B . HeLa cells were transiently transfected with murine TMEM192. Cells were treated overnight with Bafilomycin (30 nM), NH 4 Cl (25 mM), Leupeptin (100 µM), E64d (40 µM), Brefeldin A (1 µg/ml) or H 2 O or DMSO as negative control. Aliquots of total cell lysates were analysed by Western blotting using the TMEM192 antibody. Due to a low processing efficiency of TMEM192 upon overexpression conditions, a longer exposure of the membrane was included to visualize the NTF and the effect of the different inhibitors on the generation of this fragment.

    Journal: Oncotarget

    Article Title: Functional characterization of the lysosomal membrane protein TMEM192 in mice

    doi: 10.18632/oncotarget.17514

    Figure Lengend Snippet: Proteolytic processing of murine TMEM192 occurs in lysosomes A . Abundance of the TMEM192 protein (closed arrow-head) and the derived TMEM192 N-terminal fragment (NTF, open arrow-head) was compared in murine embryonic fibroblasts (MEF), RAW 264.7 cells, N2A cells and primary Bone-marrow-derived macrophages from wild type mice ( +/+ ). As a control for antibody specificity, macrophages from TMEM192 -/- were included in the analysis. Total cell lysates of the depicted cell lines were subjected to Western blotting using the TMEM192 antibody. Equal loading was confirmed by re-probing the membrane with anti-GAPDH antibody. B . HeLa cells were transiently transfected with murine TMEM192. Cells were treated overnight with Bafilomycin (30 nM), NH 4 Cl (25 mM), Leupeptin (100 µM), E64d (40 µM), Brefeldin A (1 µg/ml) or H 2 O or DMSO as negative control. Aliquots of total cell lysates were analysed by Western blotting using the TMEM192 antibody. Due to a low processing efficiency of TMEM192 upon overexpression conditions, a longer exposure of the membrane was included to visualize the NTF and the effect of the different inhibitors on the generation of this fragment.

    Article Snippet: To analyse the requirements for TMEM192 processing, the following compounds and inhibitors were employed: Bafilomycin A1 (Sigma), NH4 Cl (Roth), Leupeptin (Sigma), E64d (Enzo), Pepstatin A (Sigma), Marimastat (Sigma), Brefeldin A (Enzo).

    Techniques: Derivative Assay, Mouse Assay, Western Blot, Transfection, Negative Control, Over Expression