brefeldin a  (Cell Signaling Technology Inc)

 
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  • 99
    Name:
    Brefeldin A
    Description:
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    Catalog Number:
    9972
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    Category:
    Activators Inhibitors
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    Structured Review

    Cell Signaling Technology Inc brefeldin a
    Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor <t>brefeldin</t> A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .
    Molecular Weight
    https://www.bioz.com/result/brefeldin a/product/Cell Signaling Technology Inc
    Average 99 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination"

    Article Title: Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination

    Journal: bioRxiv

    doi: 10.1101/2020.01.21.906073

    Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .
    Figure Legend Snippet: Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Expressing, In Situ Hybridization, Immunohistochemistry

    2) Product Images from "Parallel signaling through IRE1α and PERK regulates pancreatic neuroendocrine tumor growth and survival"

    Article Title: Parallel signaling through IRE1α and PERK regulates pancreatic neuroendocrine tumor growth and survival

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-19-1116

    CRISPR/Cas9-mediated knockout of IRE1α or PERK pathways dramatically decreases INS-1 tumor burden. A, Cultured INS-1 (control) cells and the indicated CRISPR/Cas9 KO clones were treated +/− 0.625 μg/mL Brefeldin A (BFA) for 3 h prior to harvest to induce ER stress and then immunoblotted with the indicated antibodies. B, INS-1 cells and the indicated KO lines were subjected to the CellTiter-Glo luminescence-based proliferation assay at 2 and 6 d after seeding. Fold change in luminescence over 96 h was calculated for each KO line and normalized to INS-1 control (n ≥ 3). C-E, NSG mice were injected s.c. with INS-1 control and one of two unique ( C ) IRE1α, ( D ) XBP1 or ( E ) PERK KO clones. Resulting tumors were harvested and weighed 4 weeks post-injection (n ≥ 5). F-H, Photos of three representative control and ( F ) IRE1α, ( G ) XBP1 or ( H ) PERK KO tumors from C-E. I, Representative IHC for Ki67 from control and indicated KO tumors 4 weeks post-injection (scale bars, 50 μm). J, Quantification of Ki67 staining in I (n ≥ 4). * P
    Figure Legend Snippet: CRISPR/Cas9-mediated knockout of IRE1α or PERK pathways dramatically decreases INS-1 tumor burden. A, Cultured INS-1 (control) cells and the indicated CRISPR/Cas9 KO clones were treated +/− 0.625 μg/mL Brefeldin A (BFA) for 3 h prior to harvest to induce ER stress and then immunoblotted with the indicated antibodies. B, INS-1 cells and the indicated KO lines were subjected to the CellTiter-Glo luminescence-based proliferation assay at 2 and 6 d after seeding. Fold change in luminescence over 96 h was calculated for each KO line and normalized to INS-1 control (n ≥ 3). C-E, NSG mice were injected s.c. with INS-1 control and one of two unique ( C ) IRE1α, ( D ) XBP1 or ( E ) PERK KO clones. Resulting tumors were harvested and weighed 4 weeks post-injection (n ≥ 5). F-H, Photos of three representative control and ( F ) IRE1α, ( G ) XBP1 or ( H ) PERK KO tumors from C-E. I, Representative IHC for Ki67 from control and indicated KO tumors 4 weeks post-injection (scale bars, 50 μm). J, Quantification of Ki67 staining in I (n ≥ 4). * P

    Techniques Used: CRISPR, Knock-Out, Cell Culture, Clone Assay, Proliferation Assay, Mouse Assay, Injection, Immunohistochemistry, Staining

    3) Product Images from "A CREB3-regulated ER-Golgi trafficking signature promotes metastatic progression in breast cancer"

    Article Title: A CREB3-regulated ER-Golgi trafficking signature promotes metastatic progression in breast cancer

    Journal: Oncogene

    doi: 10.1038/s41388-017-0023-0

    Up-regulation of ER-Golgi trafficking and sensitization to brefeldin A in L1P and L2P cells (a) Schematic of RUSH trafficking reporter system (left panel). Immunofluorescence images show localization of ST-SBP-GFP reporter at the ER or Golgi 15 min following biotin treatment in L1P cells. GM130 was used as a Golgi marker in this experiment, scale bar: 10µm. (b) Graphs of ST-SBP-GFP and ManII-SBP-GFP reporter localization 15 min following biotin treatment (n ≥ 15 cells per sample). (c) Secreted levels of Gaussia luciferase (n=6) and alkaline phosphatase (n=5) in E1KD, M1P, L1P and L2P cells and L2P cells treated with brefeldin A (* P
    Figure Legend Snippet: Up-regulation of ER-Golgi trafficking and sensitization to brefeldin A in L1P and L2P cells (a) Schematic of RUSH trafficking reporter system (left panel). Immunofluorescence images show localization of ST-SBP-GFP reporter at the ER or Golgi 15 min following biotin treatment in L1P cells. GM130 was used as a Golgi marker in this experiment, scale bar: 10µm. (b) Graphs of ST-SBP-GFP and ManII-SBP-GFP reporter localization 15 min following biotin treatment (n ≥ 15 cells per sample). (c) Secreted levels of Gaussia luciferase (n=6) and alkaline phosphatase (n=5) in E1KD, M1P, L1P and L2P cells and L2P cells treated with brefeldin A (* P

    Techniques Used: Immunofluorescence, Marker, Luciferase

    4) Product Images from "Monoubiquitination of Cancer Stem Cell Marker CD133 at Lysine 848 Regulates Its Secretion and Promotes Cell Migration"

    Article Title: Monoubiquitination of Cancer Stem Cell Marker CD133 at Lysine 848 Regulates Its Secretion and Promotes Cell Migration

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00024-18

    K848R mutant inhibits CD133 release into EVs. (A) EVs were isolated from the medium of CD133-expressing U87MG cells and verified by transmission electron microscopy. (B) CD133 release into the supernatant was blocked following treatment with 20 μM brefeldin A. (C) Western blotting of sucrose gradient fractions confirmed the presence of CD133 together with the EV marker Tsg101 in purified EVs. (D) EVs were resuspended in PBS alone or in PBS containing 1% Triton X-100 and lysed for 2 h, followed by centrifugation for 1 h at 100,000 ×  g . The resulting supernatants (S) and pellets (P) were analyzed by Western blotting. (E to G) U87MG cells were infected with a lentivirus expressing CD133-WT or CD133-K848R. (E) EVs were verified by Western blotting of CD133 and known vesicular proteins (Hsp90 and Tsg101). A nonvesicular protein from the Golgi apparatus (GM130) was used as a control. Both cell lysates and EVs were harvested from 5 × 10 6  cells. (F) Quantification of EV CD133 relative to cell lysate CD133. Data are presented as means and SD for results from three independent experiments. Statistical analysis was performed using the Student  t  test ( * *,  P
    Figure Legend Snippet: K848R mutant inhibits CD133 release into EVs. (A) EVs were isolated from the medium of CD133-expressing U87MG cells and verified by transmission electron microscopy. (B) CD133 release into the supernatant was blocked following treatment with 20 μM brefeldin A. (C) Western blotting of sucrose gradient fractions confirmed the presence of CD133 together with the EV marker Tsg101 in purified EVs. (D) EVs were resuspended in PBS alone or in PBS containing 1% Triton X-100 and lysed for 2 h, followed by centrifugation for 1 h at 100,000 × g . The resulting supernatants (S) and pellets (P) were analyzed by Western blotting. (E to G) U87MG cells were infected with a lentivirus expressing CD133-WT or CD133-K848R. (E) EVs were verified by Western blotting of CD133 and known vesicular proteins (Hsp90 and Tsg101). A nonvesicular protein from the Golgi apparatus (GM130) was used as a control. Both cell lysates and EVs were harvested from 5 × 10 6 cells. (F) Quantification of EV CD133 relative to cell lysate CD133. Data are presented as means and SD for results from three independent experiments. Statistical analysis was performed using the Student t test ( * *, P

    Techniques Used: Mutagenesis, Isolation, Expressing, Transmission Assay, Electron Microscopy, Western Blot, Marker, Purification, Centrifugation, Infection

    5) Product Images from "Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression"

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b02073

    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Figure Legend Snippet: Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Techniques Used: Expressing, Western Blot

    6) Product Images from "The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells"

    Article Title: The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2017.00167

    Treatment with manuka honey (MH), but not sugar control (SC) solution, inhibits interleukin-6 (IL-6) secretion and production in breast cancer cells. (A) Western blot analysis of IL-6 synthesis. MDA-MB-231 cells were incubated in the presence or absence of MH (1.25 or 5%) for 4.5 h, with Brefeldin A added for the last 4 h of culture. Extracts were run on 10% SDS-PAGE and immunoblotted with a mAb specific to IL-6. The numbers below the blot indicate changes in band intensity, as determined by densitometric analysis. The data are representative of two independent experiments. (B–E) Analysis of IL-6 secretion in MDA-MB-231 (B–D) and MCF-7 (E) cells by ELISA. MDA-MB-231 cells were cultured for 12 h with or without the indicated concentrations of MH (B) or exposed to 5% MH for 2 or 4 h (C) . In a separate experiment, cells were cultured with indicated concentrations of MH or SC solution for 4 h (D) . Similarly, MCF-7 cells were cultured with the indicated MH concentrations for 4 h (E) . After culture, cell-free supernatants were collected and analyzed for IL-6 content by ELISA. The data are representative of four (MDA-MB-231 cells) or two (MCF-7 cells) independent experiments. Asterisks denote statistically significant differences in IL-6 levels of experimental groups compared to control (* p
    Figure Legend Snippet: Treatment with manuka honey (MH), but not sugar control (SC) solution, inhibits interleukin-6 (IL-6) secretion and production in breast cancer cells. (A) Western blot analysis of IL-6 synthesis. MDA-MB-231 cells were incubated in the presence or absence of MH (1.25 or 5%) for 4.5 h, with Brefeldin A added for the last 4 h of culture. Extracts were run on 10% SDS-PAGE and immunoblotted with a mAb specific to IL-6. The numbers below the blot indicate changes in band intensity, as determined by densitometric analysis. The data are representative of two independent experiments. (B–E) Analysis of IL-6 secretion in MDA-MB-231 (B–D) and MCF-7 (E) cells by ELISA. MDA-MB-231 cells were cultured for 12 h with or without the indicated concentrations of MH (B) or exposed to 5% MH for 2 or 4 h (C) . In a separate experiment, cells were cultured with indicated concentrations of MH or SC solution for 4 h (D) . Similarly, MCF-7 cells were cultured with the indicated MH concentrations for 4 h (E) . After culture, cell-free supernatants were collected and analyzed for IL-6 content by ELISA. The data are representative of four (MDA-MB-231 cells) or two (MCF-7 cells) independent experiments. Asterisks denote statistically significant differences in IL-6 levels of experimental groups compared to control (* p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Incubation, SDS Page, Enzyme-linked Immunosorbent Assay, Cell Culture

    7) Product Images from "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity"

    Article Title: Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00221.2017

    Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.
    Figure Legend Snippet: Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.

    Techniques Used: Mutagenesis

    8) Product Images from "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity"

    Article Title: Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00221.2017

    Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.
    Figure Legend Snippet: Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.

    Techniques Used: Mutagenesis

    9) Product Images from "Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer"

    Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.10.027

    Defective secretion of mature GDF11 in TNBC is caused by deficiency in PC5A convertase (A) Focal 1E6 immunoreactivity in formalin-fixed, paraffin-embedded pellets of MDA-MB-231 cells. (B) Ectopic overexpression (o.e.) of V5-tagged GDF11 in MDA-MB-231 cells. Cell extracts were immunoblotted for V5 (upper) and GDF11 (middle) with vinculin and GAPDH included as loading controls. (C) Ectopic proGDF11 expression does not phenocopy the 3D morphological changes induced by recombinant GDF11 in MDA-MB-231 cells. 3D cultures were assessed at day 12. Control cells express luciferase-V5. (D) Protein folding stress blocks the secretion of mature GDF11. MCF10A-5E cells were treated with 100 ng/ml thapsigargin (Thap), 5 μg/ml tunicamycin (Tunic), or 10 μg/ml brefeldin A (Bref) for 24 hours. ). (F and G) Maturation of proGDF11-V5 by ectopic expression of PC5A in TNBC cell lines. Cells were transduced with PC5A-V5 or LacZ-2×V5 control (F) and mature GDF11-V5 released was quantified relative to MCF10A-5E control cells (G). Quantification of proGDF11-V5 processing is shown for all cell lines used in (E). (H) High ID2 abundance is associated with compensatory PCSK5 ). Data are shown as the mean ± SEM of n = 4 (C) or 3 (E) biological replicates or the median ± 90% nonparametric confidence interval of n = 14 (H, left) or 12 (H, right) cases. For (D) to (F), conditioned medium from cell lines expressing proGDF11-V5 was V5 immunoprecipitated and immunoblotted along with cell extracts for the indicated proteins. Vinculin, Hsp90, tubulin, p38, and GAPDH were used as loading controls, and representative immunoblots are shown from biological duplicates. Scale bar is 40 μm (A) and 200 μm (C). n.s., not significant ( p > 0.05 by two-sided Ward’s test.) .
    Figure Legend Snippet: Defective secretion of mature GDF11 in TNBC is caused by deficiency in PC5A convertase (A) Focal 1E6 immunoreactivity in formalin-fixed, paraffin-embedded pellets of MDA-MB-231 cells. (B) Ectopic overexpression (o.e.) of V5-tagged GDF11 in MDA-MB-231 cells. Cell extracts were immunoblotted for V5 (upper) and GDF11 (middle) with vinculin and GAPDH included as loading controls. (C) Ectopic proGDF11 expression does not phenocopy the 3D morphological changes induced by recombinant GDF11 in MDA-MB-231 cells. 3D cultures were assessed at day 12. Control cells express luciferase-V5. (D) Protein folding stress blocks the secretion of mature GDF11. MCF10A-5E cells were treated with 100 ng/ml thapsigargin (Thap), 5 μg/ml tunicamycin (Tunic), or 10 μg/ml brefeldin A (Bref) for 24 hours. ). (F and G) Maturation of proGDF11-V5 by ectopic expression of PC5A in TNBC cell lines. Cells were transduced with PC5A-V5 or LacZ-2×V5 control (F) and mature GDF11-V5 released was quantified relative to MCF10A-5E control cells (G). Quantification of proGDF11-V5 processing is shown for all cell lines used in (E). (H) High ID2 abundance is associated with compensatory PCSK5 ). Data are shown as the mean ± SEM of n = 4 (C) or 3 (E) biological replicates or the median ± 90% nonparametric confidence interval of n = 14 (H, left) or 12 (H, right) cases. For (D) to (F), conditioned medium from cell lines expressing proGDF11-V5 was V5 immunoprecipitated and immunoblotted along with cell extracts for the indicated proteins. Vinculin, Hsp90, tubulin, p38, and GAPDH were used as loading controls, and representative immunoblots are shown from biological duplicates. Scale bar is 40 μm (A) and 200 μm (C). n.s., not significant ( p > 0.05 by two-sided Ward’s test.) .

    Techniques Used: Formalin-fixed Paraffin-Embedded, Multiple Displacement Amplification, Over Expression, Expressing, Recombinant, Luciferase, Transduction, Immunoprecipitation, Western Blot

    10) Product Images from "Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression"

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b02073

    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Figure Legend Snippet: Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Techniques Used: Expressing, Western Blot

    11) Product Images from "The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells"

    Article Title: The IL-6/STAT3 Signaling Pathway Is an Early Target of Manuka Honey-Induced Suppression of Human Breast Cancer Cells

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2017.00167

    Treatment with manuka honey (MH), but not sugar control (SC) solution, inhibits interleukin-6 (IL-6) secretion and production in breast cancer cells. (A) Western blot analysis of IL-6 synthesis. MDA-MB-231 cells were incubated in the presence or absence of MH (1.25 or 5%) for 4.5 h, with Brefeldin A added for the last 4 h of culture. Extracts were run on 10% SDS-PAGE and immunoblotted with a mAb specific to IL-6. The numbers below the blot indicate changes in band intensity, as determined by densitometric analysis. The data are representative of two independent experiments. (B–E) Analysis of IL-6 secretion in MDA-MB-231 (B–D) and MCF-7 (E) cells by ELISA. MDA-MB-231 cells were cultured for 12 h with or without the indicated concentrations of MH (B) or exposed to 5% MH for 2 or 4 h (C) . In a separate experiment, cells were cultured with indicated concentrations of MH or SC solution for 4 h (D) . Similarly, MCF-7 cells were cultured with the indicated MH concentrations for 4 h (E) . After culture, cell-free supernatants were collected and analyzed for IL-6 content by ELISA. The data are representative of four (MDA-MB-231 cells) or two (MCF-7 cells) independent experiments. Asterisks denote statistically significant differences in IL-6 levels of experimental groups compared to control (* p
    Figure Legend Snippet: Treatment with manuka honey (MH), but not sugar control (SC) solution, inhibits interleukin-6 (IL-6) secretion and production in breast cancer cells. (A) Western blot analysis of IL-6 synthesis. MDA-MB-231 cells were incubated in the presence or absence of MH (1.25 or 5%) for 4.5 h, with Brefeldin A added for the last 4 h of culture. Extracts were run on 10% SDS-PAGE and immunoblotted with a mAb specific to IL-6. The numbers below the blot indicate changes in band intensity, as determined by densitometric analysis. The data are representative of two independent experiments. (B–E) Analysis of IL-6 secretion in MDA-MB-231 (B–D) and MCF-7 (E) cells by ELISA. MDA-MB-231 cells were cultured for 12 h with or without the indicated concentrations of MH (B) or exposed to 5% MH for 2 or 4 h (C) . In a separate experiment, cells were cultured with indicated concentrations of MH or SC solution for 4 h (D) . Similarly, MCF-7 cells were cultured with the indicated MH concentrations for 4 h (E) . After culture, cell-free supernatants were collected and analyzed for IL-6 content by ELISA. The data are representative of four (MDA-MB-231 cells) or two (MCF-7 cells) independent experiments. Asterisks denote statistically significant differences in IL-6 levels of experimental groups compared to control (* p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Incubation, SDS Page, Enzyme-linked Immunosorbent Assay, Cell Culture

    12) Product Images from "CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase"

    Article Title: CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase

    Journal: Nature

    doi: 10.1038/nature15510

    Detection of GlyRS proteins in the cell medium a, c, e , Western-blot analysis of the GlyRS protein levels in NSC34 motor neurons ( a ), C2C12 cell-differentiated myotubes ( c ) and undifferentiated C2C12 myoblasts ( e ). The level of GlyRS proteins in cell medium is diminished by application of the exosome-pathway inhibitor GW4869, but not by Brefeldin A (BFA), an inhibitor of the classical endoplasmic reticulum (ER) to Golgi secretory pathway. GAPDH (cytoplasmic protein), vWF (secretory protein through ER-Golgi pathway) and TSG101 (Exosomal protein) are used as controls. b, d , Quantification of GlyRS protein level indicated in a c . Data are presented as the mean ± SEM of three independent experiments (*p
    Figure Legend Snippet: Detection of GlyRS proteins in the cell medium a, c, e , Western-blot analysis of the GlyRS protein levels in NSC34 motor neurons ( a ), C2C12 cell-differentiated myotubes ( c ) and undifferentiated C2C12 myoblasts ( e ). The level of GlyRS proteins in cell medium is diminished by application of the exosome-pathway inhibitor GW4869, but not by Brefeldin A (BFA), an inhibitor of the classical endoplasmic reticulum (ER) to Golgi secretory pathway. GAPDH (cytoplasmic protein), vWF (secretory protein through ER-Golgi pathway) and TSG101 (Exosomal protein) are used as controls. b, d , Quantification of GlyRS protein level indicated in a c . Data are presented as the mean ± SEM of three independent experiments (*p

    Techniques Used: Western Blot

    13) Product Images from "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity"

    Article Title: Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00221.2017

    Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.
    Figure Legend Snippet: Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.

    Techniques Used: Mutagenesis

    14) Product Images from "Apolipoprotein L9 interacts with LC3/GABARAP and is a microtubule-associated protein with a widespread subcellular distribution"

    Article Title: Apolipoprotein L9 interacts with LC3/GABARAP and is a microtubule-associated protein with a widespread subcellular distribution

    Journal: bioRxiv

    doi: 10.1101/671065

    Subcellular distribution of ApoL9 after treatment with bafilomycin A1 and brefeldin A A. Patterns of distribution of ApoL9-V5 in various cells of B16F10 L9 after treatment with 200 nM bafilomycin A1 for 10-18 h, showing: ring-shaped structures (1), accumulation in the juxtanuclear region (2), and curved, thread-like structures (3, 4). Anti-V5 antibody was used to detect ApoL9. B. Indirect immunofluorescence for ApoL-V5 and lysosomal marker LAMP1 in B16F10 L9 cells treated with bafilomycin A1 for 12 h, showing co-localization in the juxtanuclear region (upper panel). ApoL9 in curved, thread-like structures (lower panel) does not co-localize with LAMP1. Anti-V5 and anti-LAMP1 antibodies were used. C. Indirect immunofluorescence for ApoL9 and outer mitochondrial membrane (OMM) marker TSPO in B16F10 L9 cells treated with bafilomycin A1 for 12 h, showing co-localization of ApoL9-V5-positive rings with TSPO-GFP (upper panel, yellow arrows). ApoL9-V5 in curved, threadlike structures (lower panel) does not co-localize with TSPO-GFP. Tspo-GFP was electroporated into B16F10 L9 cells. D . Patterns of distribution of ApoL9-V5 in various cells after treatment with 10 mg/mL brefeldin A for 14-18 h. Note the proximity between ring-shaped structures (yellow arrows) in images of higher magnification. E. Indirect immunofluorescence for ApoL9-V5 and TSPO-GFP in B16F10 L9 cells treated with brefeldin A, and (right) magnified inset. Note that only a fraction of the mitochondria are positive for ApoL9. F . A B16F10 L9 cell electroporated with the GFP-Lc3b construct and treated with bafilomycin A1 for 12 h, showing GFP-LC3B on punctate autophagosomes. ApoL9-V5 is not present on autophagosomes, but partial co-localization is visible in a few places (magnified boxed inset; white arrows). G-H. Images showing GFP-LC3B (transfected by electroporation) in the vicinity of ApoL9-containing mitochondria in B16F10 L9 cells treated with bafilomycin A1 and brefeldin A, respectively. Boxed insets are magnified. I. An ApoL9-positive ring-shaped vesicle exiting the cell boundary. B16F10 L9 cells were treated with bafilomycin A1 for 16h.
    Figure Legend Snippet: Subcellular distribution of ApoL9 after treatment with bafilomycin A1 and brefeldin A A. Patterns of distribution of ApoL9-V5 in various cells of B16F10 L9 after treatment with 200 nM bafilomycin A1 for 10-18 h, showing: ring-shaped structures (1), accumulation in the juxtanuclear region (2), and curved, thread-like structures (3, 4). Anti-V5 antibody was used to detect ApoL9. B. Indirect immunofluorescence for ApoL-V5 and lysosomal marker LAMP1 in B16F10 L9 cells treated with bafilomycin A1 for 12 h, showing co-localization in the juxtanuclear region (upper panel). ApoL9 in curved, thread-like structures (lower panel) does not co-localize with LAMP1. Anti-V5 and anti-LAMP1 antibodies were used. C. Indirect immunofluorescence for ApoL9 and outer mitochondrial membrane (OMM) marker TSPO in B16F10 L9 cells treated with bafilomycin A1 for 12 h, showing co-localization of ApoL9-V5-positive rings with TSPO-GFP (upper panel, yellow arrows). ApoL9-V5 in curved, threadlike structures (lower panel) does not co-localize with TSPO-GFP. Tspo-GFP was electroporated into B16F10 L9 cells. D . Patterns of distribution of ApoL9-V5 in various cells after treatment with 10 mg/mL brefeldin A for 14-18 h. Note the proximity between ring-shaped structures (yellow arrows) in images of higher magnification. E. Indirect immunofluorescence for ApoL9-V5 and TSPO-GFP in B16F10 L9 cells treated with brefeldin A, and (right) magnified inset. Note that only a fraction of the mitochondria are positive for ApoL9. F . A B16F10 L9 cell electroporated with the GFP-Lc3b construct and treated with bafilomycin A1 for 12 h, showing GFP-LC3B on punctate autophagosomes. ApoL9-V5 is not present on autophagosomes, but partial co-localization is visible in a few places (magnified boxed inset; white arrows). G-H. Images showing GFP-LC3B (transfected by electroporation) in the vicinity of ApoL9-containing mitochondria in B16F10 L9 cells treated with bafilomycin A1 and brefeldin A, respectively. Boxed insets are magnified. I. An ApoL9-positive ring-shaped vesicle exiting the cell boundary. B16F10 L9 cells were treated with bafilomycin A1 for 16h.

    Techniques Used: Immunofluorescence, Marker, Construct, Transfection, Electroporation

    Effect of treatment with various compounds on ApoL9 protein levels in the cell A. Immunoblotting for ApoL9-V5 in Triton X-100-soluble and insoluble fractions of B16F10 L9 cells treated with bafilomycin A1 for the indicated time periods, and (below) quantification of the blots. B. Immunoblotting for ApoL9-V5 in the Triton X-100-soluble fraction of B16F10 L9 cells treated with brefeldin A for the indicated time periods and (below) quantification. No ApoL9 was detected in the Triton X-100-insoluble fraction. C. Immunoblotting for endogenous ApoL9 in hepatoma H6 treated with brefeldin A for the indicated time periods and (below) quantification. Anti-MBP-ApoL9 antibody was used to detect ApoL9. D . qPCR for Apol9 mRNA levels in hepatoma H6 treated with brefeldin A for the same time periods as in C., normalized to Gapdh. Error bars indicate mean ± s.d (n=3). E-F. Immunoblotting for ApoL9-V5 in B16F10 L9 cells treated with EBSS alone, or EBSS+ 400 nM bafilomycin A1, for the indicated time periods. Blots were stripped and reprobed with anti-LC3B antibody to confirm the effect of autophagy inhibition. Blots are quantified and represented as graphs (right). G . Immunoblotting for ApoL9-V5 in B16F10 L9 cells after treatment with 50 mg/mL cycloheximide (CHX) for the indicated time periods and (far right) quantification of the blot. The blot is stained with Ponceau S to show loading.
    Figure Legend Snippet: Effect of treatment with various compounds on ApoL9 protein levels in the cell A. Immunoblotting for ApoL9-V5 in Triton X-100-soluble and insoluble fractions of B16F10 L9 cells treated with bafilomycin A1 for the indicated time periods, and (below) quantification of the blots. B. Immunoblotting for ApoL9-V5 in the Triton X-100-soluble fraction of B16F10 L9 cells treated with brefeldin A for the indicated time periods and (below) quantification. No ApoL9 was detected in the Triton X-100-insoluble fraction. C. Immunoblotting for endogenous ApoL9 in hepatoma H6 treated with brefeldin A for the indicated time periods and (below) quantification. Anti-MBP-ApoL9 antibody was used to detect ApoL9. D . qPCR for Apol9 mRNA levels in hepatoma H6 treated with brefeldin A for the same time periods as in C., normalized to Gapdh. Error bars indicate mean ± s.d (n=3). E-F. Immunoblotting for ApoL9-V5 in B16F10 L9 cells treated with EBSS alone, or EBSS+ 400 nM bafilomycin A1, for the indicated time periods. Blots were stripped and reprobed with anti-LC3B antibody to confirm the effect of autophagy inhibition. Blots are quantified and represented as graphs (right). G . Immunoblotting for ApoL9-V5 in B16F10 L9 cells after treatment with 50 mg/mL cycloheximide (CHX) for the indicated time periods and (far right) quantification of the blot. The blot is stained with Ponceau S to show loading.

    Techniques Used: Real-time Polymerase Chain Reaction, Inhibition, Staining

    Properties of the region(s) of ApoL9 essential for PE-binding A . Protein-lipid overlay assay for screening PE-binding in MBP-ApoL9 deletion mutants. 1 mg of dipalmitoyl-PE was spotted in quadruplicate on nitrocellulose membrane. Red asterisks indicate mutants that failed to bind PE. B. Immunostaining for ApoL9-V5 after lipofection of the insert-less vector pBApo-EF1 α pur into cell lines stably expressing wild type ApoL9 or ApoL9 transmembrane deletion mutants for 4 h. Note the characteristic DOPE-induced aggregation only in WT. C, D . Subcellular distribution pattern of ApoL9Δ111-123 and ApoL9Δ124-144 electroporated into B16F10 cells and treated with 10 mg/mL brefeldin A for 18 h, or 10 mM MG132 for 6 h. Note the difference in pattern after MG132 treatment. E. Indirect immunofluorescence for ApoL9-V5 and LAMP1 in B16F10 L9Δ111-123 and B16F10 L9Δ124-144 after treatment with 200 nM bafilomycin A1 for 16 h. Boxed insets are magnified for better viewing.
    Figure Legend Snippet: Properties of the region(s) of ApoL9 essential for PE-binding A . Protein-lipid overlay assay for screening PE-binding in MBP-ApoL9 deletion mutants. 1 mg of dipalmitoyl-PE was spotted in quadruplicate on nitrocellulose membrane. Red asterisks indicate mutants that failed to bind PE. B. Immunostaining for ApoL9-V5 after lipofection of the insert-less vector pBApo-EF1 α pur into cell lines stably expressing wild type ApoL9 or ApoL9 transmembrane deletion mutants for 4 h. Note the characteristic DOPE-induced aggregation only in WT. C, D . Subcellular distribution pattern of ApoL9Δ111-123 and ApoL9Δ124-144 electroporated into B16F10 cells and treated with 10 mg/mL brefeldin A for 18 h, or 10 mM MG132 for 6 h. Note the difference in pattern after MG132 treatment. E. Indirect immunofluorescence for ApoL9-V5 and LAMP1 in B16F10 L9Δ111-123 and B16F10 L9Δ124-144 after treatment with 200 nM bafilomycin A1 for 16 h. Boxed insets are magnified for better viewing.

    Techniques Used: Binding Assay, Protein-lipid Overlay Assay (PLOA), Immunostaining, Plasmid Preparation, Stable Transfection, Expressing, Immunofluorescence

    15) Product Images from "The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment *"

    Article Title: The Ubiquitin Regulatory X (UBX) Domain-containing Protein TUG Regulates the p97 ATPase and Resides at the Endoplasmic Reticulum-Golgi Intermediate Compartment *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284232

    TUG depletion alters GM130 staining intensity and impairs Golgi reformation after brefeldin A washout. A , endogenous TUG and GM130 were imaged using immunofluorescence and confocal microscopy of HeLa cells. Cells were treated with control siRNA, TUG siRNA
    Figure Legend Snippet: TUG depletion alters GM130 staining intensity and impairs Golgi reformation after brefeldin A washout. A , endogenous TUG and GM130 were imaged using immunofluorescence and confocal microscopy of HeLa cells. Cells were treated with control siRNA, TUG siRNA

    Techniques Used: Staining, Immunofluorescence, Confocal Microscopy

    16) Product Images from "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity"

    Article Title: Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00221.2017

    Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.
    Figure Legend Snippet: Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.

    Techniques Used: Mutagenesis

    17) Product Images from "Disparate Bone Anabolic Cues Activate Bone Formation by Regulating the Rapid Lysosomal Degradation of Sclerostin Protein"

    Article Title: Disparate Bone Anabolic Cues Activate Bone Formation by Regulating the Rapid Lysosomal Degradation of Sclerostin Protein

    Journal: bioRxiv

    doi: 10.1101/2020.10.26.355800

    Sclerostin is rapidly degraded by the lysosome following bone anabolic stimuli. (A)  Ocy454 cells transfected with GFP-sclerostin were treated with cycloheximide (150µg/mL) and either DMSO (0.1%), Bafilomycin A1 (100nM), Brefeldin A (2μm), or MG-132 (10μm) 4 hours prior to FSS. Cells were subjected to 5 minutes of FSS at 4 dynes/cm 2  and lysed immediately after the end of FSS or 30 minutes after the conclusion of FSS. Western blots were probed for sclerostin and β-actin. Timecourse shows mean ± SEM (n=3-6 independent experiments/group). B)Ocy454 cells transfected with GFP-sclerostin were pre-treated with DMSO (0.1%) or Bafilomycin A1 (100nM) for 30 minutes prior to the addition of vehicle or PTH (  1 -  34 ) (10nM) for an additional 30 minutes (n=3). Sclerostin abundance relative to the loading control was quantified. Graph depicts mean ± SD. *p
    Figure Legend Snippet: Sclerostin is rapidly degraded by the lysosome following bone anabolic stimuli. (A) Ocy454 cells transfected with GFP-sclerostin were treated with cycloheximide (150µg/mL) and either DMSO (0.1%), Bafilomycin A1 (100nM), Brefeldin A (2μm), or MG-132 (10μm) 4 hours prior to FSS. Cells were subjected to 5 minutes of FSS at 4 dynes/cm 2 and lysed immediately after the end of FSS or 30 minutes after the conclusion of FSS. Western blots were probed for sclerostin and β-actin. Timecourse shows mean ± SEM (n=3-6 independent experiments/group). B)Ocy454 cells transfected with GFP-sclerostin were pre-treated with DMSO (0.1%) or Bafilomycin A1 (100nM) for 30 minutes prior to the addition of vehicle or PTH ( 1 - 34 ) (10nM) for an additional 30 minutes (n=3). Sclerostin abundance relative to the loading control was quantified. Graph depicts mean ± SD. *p

    Techniques Used: Transfection, Western Blot

    18) Product Images from "Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression"

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b02073

    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Figure Legend Snippet: Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Techniques Used: Expressing, Western Blot

    19) Product Images from "Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination"

    Article Title: Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination

    Journal: bioRxiv

    doi: 10.1101/2020.01.21.906073

    Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .
    Figure Legend Snippet: Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Expressing, In Situ Hybridization, Immunohistochemistry

    20) Product Images from "A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila"

    Article Title: A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.109975

    Serpentine accumulation in the fat body cells of vesicle transport mutants and drug-treated embryos. (A-C) Stage 16 control (A), rab9 (B) and shrub (C) homozygous mutant embryos stained with anti-Serp and anti-Seven-up (Svp). Yellow arrowheads in B and C indicate strong Serp accumulation in the fat body cells. (D,E) Stage 16 control and ppl- GAL4- shi K44A-expressing embryos stained with anti-Serp and anti-Svp. Yellow arrowhead in E indicates Serp accumulation in Svp-positive cells. (F,F′) Embryos treated with DMSO (top panel) and brefeldin A (BFA) (bottom panel). BFA caused substantial amounts of Serp to be retained within the fat body cells (yellow arrowheads in F and F′). (G) Stage 16 svp 1 mutant embryos stained with anti-Serp antibody (top) and counter staining with DAPI (bottom). Yellow arrowhead indicates Serp accumulation in mesodermal cells. Scale bars: 10 μm.
    Figure Legend Snippet: Serpentine accumulation in the fat body cells of vesicle transport mutants and drug-treated embryos. (A-C) Stage 16 control (A), rab9 (B) and shrub (C) homozygous mutant embryos stained with anti-Serp and anti-Seven-up (Svp). Yellow arrowheads in B and C indicate strong Serp accumulation in the fat body cells. (D,E) Stage 16 control and ppl- GAL4- shi K44A-expressing embryos stained with anti-Serp and anti-Svp. Yellow arrowhead in E indicates Serp accumulation in Svp-positive cells. (F,F′) Embryos treated with DMSO (top panel) and brefeldin A (BFA) (bottom panel). BFA caused substantial amounts of Serp to be retained within the fat body cells (yellow arrowheads in F and F′). (G) Stage 16 svp 1 mutant embryos stained with anti-Serp antibody (top) and counter staining with DAPI (bottom). Yellow arrowhead indicates Serp accumulation in mesodermal cells. Scale bars: 10 μm.

    Techniques Used: Mutagenesis, Staining, Expressing

    21) Product Images from "Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression"

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b02073

    Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.
    Figure Legend Snippet: Similar APP expression pattern by brefeldin A, a protein ER-to-Golgi traffic blocker. (A) Chemical structures of isoginkgetin and brefeldin A. (B) Western blots of time-coursed APP expression in HEK293T cells treated with 30 μM IGK, 5 μM brefeldin A, and DMSO.

    Techniques Used: Expressing, Western Blot

    22) Product Images from "A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila"

    Article Title: A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.109975

    Serpentine accumulation in the fat body cells of vesicle transport mutants and drug-treated embryos. (A-C) Stage 16 control (A), rab9 (B) and shrub (C) homozygous mutant embryos stained with anti-Serp and anti-Seven-up (Svp). Yellow arrowheads in B and C indicate strong Serp accumulation in the fat body cells. (D,E) Stage 16 control and ppl- GAL4- shi K44A-expressing embryos stained with anti-Serp and anti-Svp. Yellow arrowhead in E indicates Serp accumulation in Svp-positive cells. (F,F′) Embryos treated with DMSO (top panel) and brefeldin A (BFA) (bottom panel). BFA caused substantial amounts of Serp to be retained within the fat body cells (yellow arrowheads in F and F′). (G) Stage 16 svp 1 mutant embryos stained with anti-Serp antibody (top) and counter staining with DAPI (bottom). Yellow arrowhead indicates Serp accumulation in mesodermal cells. Scale bars: 10 μm.
    Figure Legend Snippet: Serpentine accumulation in the fat body cells of vesicle transport mutants and drug-treated embryos. (A-C) Stage 16 control (A), rab9 (B) and shrub (C) homozygous mutant embryos stained with anti-Serp and anti-Seven-up (Svp). Yellow arrowheads in B and C indicate strong Serp accumulation in the fat body cells. (D,E) Stage 16 control and ppl- GAL4- shi K44A-expressing embryos stained with anti-Serp and anti-Svp. Yellow arrowhead in E indicates Serp accumulation in Svp-positive cells. (F,F′) Embryos treated with DMSO (top panel) and brefeldin A (BFA) (bottom panel). BFA caused substantial amounts of Serp to be retained within the fat body cells (yellow arrowheads in F and F′). (G) Stage 16 svp 1 mutant embryos stained with anti-Serp antibody (top) and counter staining with DAPI (bottom). Yellow arrowhead indicates Serp accumulation in mesodermal cells. Scale bars: 10 μm.

    Techniques Used: Mutagenesis, Staining, Expressing

    23) Product Images from "PRDM14 is overexpressed in chronic pancreatitis prior to pancreatic cancer"

    Article Title: PRDM14 is overexpressed in chronic pancreatitis prior to pancreatic cancer

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12519

    PRDM 14 expression in pancreatic cancer cell lines. Pancreatic cancer cell lines,  PK ‐1 and As PC ‐1, were treated with cerulein and  ER  stress inducer brefeldin A. Expression levels of  PRDM 14,  GAPDH ,  GRP 78,  eIF 2α, phospho‐ eIF 2α, and  CHOP  in the cells were analyzed using Wes capillary electrophoresis system.  GAPDH  was used as a loading control. Expression levels of  PRDM 14 protein were normalized to that of  GAPDH  and plotted as fold changes relative to control. Error bars represent the mean ±  SD  of triplicate samples. Student's  t ‐test: ** P
    Figure Legend Snippet: PRDM 14 expression in pancreatic cancer cell lines. Pancreatic cancer cell lines, PK ‐1 and As PC ‐1, were treated with cerulein and ER stress inducer brefeldin A. Expression levels of PRDM 14, GAPDH , GRP 78, eIF 2α, phospho‐ eIF 2α, and CHOP in the cells were analyzed using Wes capillary electrophoresis system. GAPDH was used as a loading control. Expression levels of PRDM 14 protein were normalized to that of GAPDH and plotted as fold changes relative to control. Error bars represent the mean ± SD of triplicate samples. Student's t ‐test: ** P

    Techniques Used: Expressing, Electrophoresis

    24) Product Images from "Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity"

    Article Title: Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00221.2017

    Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.
    Figure Legend Snippet: Mutations in HDS2 (homology downstream of Sec7 domain 2) of Golgi brefeldin A-resistant factor 1 (GBF1) analyzed in this study. A : schematic representation of full-length GBF1 showing overall domain organization and location of mutations within the HDS2 domain analyzed in this study. The A795E mutation within the Sec7d that confers brefeldin A (BFA) resistance is indicated. B : HDS2 sequences for Homo sapiens GBF1 ( Hsap .1) and GBF1 orthologs from Danio rerio ( Drer .1) and Drosophila melanogaster ( Dmel .1) retrieved from GenBank were aligned using Clustal Omega. Identical amino acids are shaded in yellow and indicated by asterisks below the sequences. Strongly similar amino acids are indicated by dark green shading and a colon below the sequences. Weakly similar amino acids are indicated by light green shading and a period below the sequences. Predicted α-helical regions are indicated by black lines above the sequences. C–F : helical wheel projections of the 4 α-helices analyzed in this study with the mutated residues indicated with asterisks. Hydrophilic residues are circles, hydrophobic residues are squares, potentially negatively charged residues are triangles, and potentially positively charged residues are pentagons. The most hydrophobic residues are green, the amount of green decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are red, with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity.

    Techniques Used: Mutagenesis

    Related Articles

    other:

    Article Title: Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination
    Article Snippet: Consistent with active secretion of sulfatases ( , ), blockade of the secretory pathway with Brefeldin A led to cytoplasmic SULF2 protein accumulation in hOPCs ( ).

    Cell Culture:

    Article Title: Noncanonical autophagy at ER exit sites regulates procollagen turnover
    Article Snippet: .. Fifty micromolar H89 (Sigma-Aldrich), 5 µg/mL BFA (Cell Signaling), 100 nM bafilomycin A1 (Sigma-Aldrich), and 100 µM leupeptin (Sigma-Aldrich) were added to the cell culture media as needed at the time points indicated in the text. .. Cells were fixed in freshly prepared methanol-free 2% formaldehyde (Thermo Fisher Scientific) solution in PBS, pH 7.4, for 10–15 min, washed in PBS, permeabilized in 0.4% Triton X in PBS for 10 min, and returned to PBS.

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    Cell Signaling Technology Inc bfa
    The effect of <t>BFA</t> on the distribution of EBV structural proteins and the release of virions. (A–E) The effect of BFA on the distribution of gp350/220 and organelle markers in <t>Akata</t> + cells induced into the lytic cycle. Akata + cells were treated with αhIgG in the presence or absence of 100 nM BFA for 16 h. The distribution of gp350/220 (green), TGN46 (A) , GM130 (B) , CD63 (C) , Rab11 (D) , and Rab27a (E) are shown. The nuclei were counterstained with Hoechst 33342. Insets show the boxed areas. Scale bars: 10 μm. (F) The effect of BFA on expression of p18 in Akata + cells undergoing the lytic cycle. Akata + cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 16 h. Total cell lysates were analyzed by Western blotting with antibodies against p18 or β-actin. (G) The effect of BFA on the release of virions. Akata - eGFP-EBV cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 48 h. Supernatants containing eGFP-EBV were harvested and incubated with Daudi - cells for 24 h. The percentages of eGFP-positive infected Daudi - cells were analyzed by means of flow cytometry. The experiment was performed three times independently and the average values and their standard deviations are shown for each condition. ∗ P
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    The effect of BFA on the distribution of EBV structural proteins and the release of virions. (A–E) The effect of BFA on the distribution of gp350/220 and organelle markers in Akata + cells induced into the lytic cycle. Akata + cells were treated with αhIgG in the presence or absence of 100 nM BFA for 16 h. The distribution of gp350/220 (green), TGN46 (A) , GM130 (B) , CD63 (C) , Rab11 (D) , and Rab27a (E) are shown. The nuclei were counterstained with Hoechst 33342. Insets show the boxed areas. Scale bars: 10 μm. (F) The effect of BFA on expression of p18 in Akata + cells undergoing the lytic cycle. Akata + cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 16 h. Total cell lysates were analyzed by Western blotting with antibodies against p18 or β-actin. (G) The effect of BFA on the release of virions. Akata - eGFP-EBV cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 48 h. Supernatants containing eGFP-EBV were harvested and incubated with Daudi - cells for 24 h. The percentages of eGFP-positive infected Daudi - cells were analyzed by means of flow cytometry. The experiment was performed three times independently and the average values and their standard deviations are shown for each condition. ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Epstein–Barr Virus Acquires Its Final Envelope on Intracellular Compartments With Golgi Markers

    doi: 10.3389/fmicb.2018.00454

    Figure Lengend Snippet: The effect of BFA on the distribution of EBV structural proteins and the release of virions. (A–E) The effect of BFA on the distribution of gp350/220 and organelle markers in Akata + cells induced into the lytic cycle. Akata + cells were treated with αhIgG in the presence or absence of 100 nM BFA for 16 h. The distribution of gp350/220 (green), TGN46 (A) , GM130 (B) , CD63 (C) , Rab11 (D) , and Rab27a (E) are shown. The nuclei were counterstained with Hoechst 33342. Insets show the boxed areas. Scale bars: 10 μm. (F) The effect of BFA on expression of p18 in Akata + cells undergoing the lytic cycle. Akata + cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 16 h. Total cell lysates were analyzed by Western blotting with antibodies against p18 or β-actin. (G) The effect of BFA on the release of virions. Akata - eGFP-EBV cells were treated with or without αhIgG in the presence or absence of 100 nM BFA for 48 h. Supernatants containing eGFP-EBV were harvested and incubated with Daudi - cells for 24 h. The percentages of eGFP-positive infected Daudi - cells were analyzed by means of flow cytometry. The experiment was performed three times independently and the average values and their standard deviations are shown for each condition. ∗ P

    Article Snippet: Brefeldin A Treatment For brefeldin A (BFA) treatment, 100 nM BFA (Cell Signaling Technology) was added to Akata+ cells (5 × 105 cells) at 6 h post-induction of the lytic cycle.

    Techniques: Expressing, Western Blot, Incubation, Infection, Flow Cytometry, Cytometry

    DTT and SubAB induce IRE1 to undergo strong and rapid phosphorylation at S729. (A) 5TGM1 cells were treated with 5 mM DTT for 3 h or with 3.5 µM BFA, 20 µM B-I09, 50 µM MG132, 1 nM SubAB, 2.5 µM Tg, or 5 µg/ml Tu for 12 h. Lysates were immunoblotted for the indicated proteins. (B) 5TGM1 cells were treated with 5 mM DTT for the indicated times and immunoblotted. (C) 5TGM1 cells were treated with DTT at the indicated concentrations for 3 h and immunoblotted. (D) 5TGM1 cells were treated with 5 mM DTT for 3 h or with 1.5 nM SubAB for 24 h and immunoblotted. (E) 5TGM1 cells were treated with 5 mM DTT for 3 h or with SubAB for 12 h using increasing concentrations and immunoblotted. (F) 5TGM1 cells were exposed to 1 nM SubAB for 12 h and were subsequently treated with KIRA6, staurosporine, imatinib, or sunitinib at 5 µM for an additional 2 h and then immunoblotted. Data in this figure are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

    doi: 10.1083/jcb.201709137

    Figure Lengend Snippet: DTT and SubAB induce IRE1 to undergo strong and rapid phosphorylation at S729. (A) 5TGM1 cells were treated with 5 mM DTT for 3 h or with 3.5 µM BFA, 20 µM B-I09, 50 µM MG132, 1 nM SubAB, 2.5 µM Tg, or 5 µg/ml Tu for 12 h. Lysates were immunoblotted for the indicated proteins. (B) 5TGM1 cells were treated with 5 mM DTT for the indicated times and immunoblotted. (C) 5TGM1 cells were treated with DTT at the indicated concentrations for 3 h and immunoblotted. (D) 5TGM1 cells were treated with 5 mM DTT for 3 h or with 1.5 nM SubAB for 24 h and immunoblotted. (E) 5TGM1 cells were treated with 5 mM DTT for 3 h or with SubAB for 12 h using increasing concentrations and immunoblotted. (F) 5TGM1 cells were exposed to 1 nM SubAB for 12 h and were subsequently treated with KIRA6, staurosporine, imatinib, or sunitinib at 5 µM for an additional 2 h and then immunoblotted. Data in this figure are representative of three independent experiments.

    Article Snippet: LPS (Sigma-Aldrich), DTT (Sigma-Aldrich), BFA (Cell Signaling Technology), CpG-1826 oligodeoxynucleotides (TIB-Molbiol), Tu (Enzo Life Sciences), Tg (Enzo Life Sciences), KIRA6 (EMD Millipore), staurosporine (EMD Millipore), imatinib (EMD Millipore), sunitinib (Sigma-Aldrich), MG-132 (Enzo Life Sciences), Z-VAD-FMK (Enzo Life Sciences), chloroquine (Cell Signaling Technology), and NH4 Cl (Sigma-Aldrich) were also purchased from commercial sources.

    Techniques:

    Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .

    Journal: bioRxiv

    Article Title: Overcoming the inhibitory microenvironment surrounding oligodendrocyte progenitor cells following demyelination

    doi: 10.1101/2020.01.21.906073

    Figure Lengend Snippet: Sulfatase is expressed by mouse and human OPCs. a , RT-PCR analysis of SULF2 mRNA in human primary OPCs during in vitro differentiation after PDGF-AA removal. b - c , SULF2 protein is expressed at low levels in hOPCs ( b ), and immunoreactivity substantially increased after treatment with the protein secretion inhibitor brefeldin A (5 µg/ml) ( c ). SULF2 mRNA expressing PDGFRA + OPCs in fetal human brain ( d ). e-g , Sulf1 and Sulf2 expression was analyzed in mouse corpus callosum during normal development by RNAscope in situ hybridization and combined with Olig2 Immunohistochemistry (IHC) at postnatal day 7 ( e, h ), day 28 ( f, i ) and at 24 weeks ( g, j ). Sulf1 mRNA (cyan), Sulf2 mRNA (green), Pdgfra mRNA (red) and Olig2 protein (pink). White arrows denote Pdgfra + OPCs expressing Sulf1 and Sulf2 . Arrowheads denote Sulf1 + Pdgfra + OPCs. Yellow arrows denote Olig2 + Pdgfra - oligodendrocytes that express Sulf2 . Scale: 100 µm ( b-c ), 20 µm ( d-j) .

    Article Snippet: Consistent with active secretion of sulfatases ( , ), blockade of the secretory pathway with Brefeldin A led to cytoplasmic SULF2 protein accumulation in hOPCs ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, In Vitro, Expressing, In Situ Hybridization, Immunohistochemistry

    CRISPR/Cas9-mediated knockout of IRE1α or PERK pathways dramatically decreases INS-1 tumor burden. A, Cultured INS-1 (control) cells and the indicated CRISPR/Cas9 KO clones were treated +/− 0.625 μg/mL Brefeldin A (BFA) for 3 h prior to harvest to induce ER stress and then immunoblotted with the indicated antibodies. B, INS-1 cells and the indicated KO lines were subjected to the CellTiter-Glo luminescence-based proliferation assay at 2 and 6 d after seeding. Fold change in luminescence over 96 h was calculated for each KO line and normalized to INS-1 control (n ≥ 3). C-E, NSG mice were injected s.c. with INS-1 control and one of two unique ( C ) IRE1α, ( D ) XBP1 or ( E ) PERK KO clones. Resulting tumors were harvested and weighed 4 weeks post-injection (n ≥ 5). F-H, Photos of three representative control and ( F ) IRE1α, ( G ) XBP1 or ( H ) PERK KO tumors from C-E. I, Representative IHC for Ki67 from control and indicated KO tumors 4 weeks post-injection (scale bars, 50 μm). J, Quantification of Ki67 staining in I (n ≥ 4). * P

    Journal: Cancer research

    Article Title: Parallel signaling through IRE1α and PERK regulates pancreatic neuroendocrine tumor growth and survival

    doi: 10.1158/0008-5472.CAN-19-1116

    Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of IRE1α or PERK pathways dramatically decreases INS-1 tumor burden. A, Cultured INS-1 (control) cells and the indicated CRISPR/Cas9 KO clones were treated +/− 0.625 μg/mL Brefeldin A (BFA) for 3 h prior to harvest to induce ER stress and then immunoblotted with the indicated antibodies. B, INS-1 cells and the indicated KO lines were subjected to the CellTiter-Glo luminescence-based proliferation assay at 2 and 6 d after seeding. Fold change in luminescence over 96 h was calculated for each KO line and normalized to INS-1 control (n ≥ 3). C-E, NSG mice were injected s.c. with INS-1 control and one of two unique ( C ) IRE1α, ( D ) XBP1 or ( E ) PERK KO clones. Resulting tumors were harvested and weighed 4 weeks post-injection (n ≥ 5). F-H, Photos of three representative control and ( F ) IRE1α, ( G ) XBP1 or ( H ) PERK KO tumors from C-E. I, Representative IHC for Ki67 from control and indicated KO tumors 4 weeks post-injection (scale bars, 50 μm). J, Quantification of Ki67 staining in I (n ≥ 4). * P

    Article Snippet: Brefeldin A (9972S) was purchased from Cell Signaling Technology; tunicamycin (T7765), thapsigargin (T9033) and doxycycline (D9891) were purchased from Sigma-Aldrich.

    Techniques: CRISPR, Knock-Out, Cell Culture, Clone Assay, Proliferation Assay, Mouse Assay, Injection, Immunohistochemistry, Staining