bafilomycin a1  (Millipore)


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    Structured Review

    Millipore bafilomycin a1
    Identification of putative ion channel defective mutants by homology modeling and <t>bafilomycin</t> A1 rescue. A) Molecular models of N-terminal region mutants that yielded > 1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). B) Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. C) Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production"

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005297

    Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue. A) Molecular models of N-terminal region mutants that yielded > 1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). B) Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. C) Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p
    Figure Legend Snippet: Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue. A) Molecular models of N-terminal region mutants that yielded > 1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). B) Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. C) Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p

    Techniques Used: Mutagenesis, Blocking Assay, Binding Assay, Electroporation, Cell Culture

    2) Product Images from "Cadmium Impairs Albumin Reabsorption by Down-regulating Megalin and ClC5 Channels in Renal Proximal Tubule Cells"

    Article Title: Cadmium Impairs Albumin Reabsorption by Down-regulating Megalin and ClC5 Channels in Renal Proximal Tubule Cells

    Journal: Environmental Health Perspectives

    doi: 10.1289/ehp.0901874

    Megalin and ClC5 undergo enhanced degradation via the lysosomal pathway. ( A ) Representative Western blots of cell lysates from LLC-PK1 cells incubated for 9 hr with 10 μM CdCl 2 , 10 μM MG-132, or 10 μM MG-132 plus 10 μM CdCl 2 . ( B and C ) Densitometric analyses of megalin ( B ) and ClC5 ( C ) protein from Western blots shown in A . ( D ) Western blots of cell lysates from LLC-PK1 cells exposed for 9 hr to 10 μM CdCl 2 , 1 μM bafilomycin A-1 (Bafilo), or 1 μM Bafilo plus 10 μM CdCl 2 . ( E and F ) Densitometric analyses of megalin ( E ) and ClC5 ( F ) protein from Western blots shown in D . Untreated controls were incubated with serum-free medium containing 0.1% DMSO, the same concentration used in treated cells. For B, C, D, and F , all values were normalized against β-actin expression, and the control values were set to 100%. Values are mean ± SE of three experiments. * p
    Figure Legend Snippet: Megalin and ClC5 undergo enhanced degradation via the lysosomal pathway. ( A ) Representative Western blots of cell lysates from LLC-PK1 cells incubated for 9 hr with 10 μM CdCl 2 , 10 μM MG-132, or 10 μM MG-132 plus 10 μM CdCl 2 . ( B and C ) Densitometric analyses of megalin ( B ) and ClC5 ( C ) protein from Western blots shown in A . ( D ) Western blots of cell lysates from LLC-PK1 cells exposed for 9 hr to 10 μM CdCl 2 , 1 μM bafilomycin A-1 (Bafilo), or 1 μM Bafilo plus 10 μM CdCl 2 . ( E and F ) Densitometric analyses of megalin ( E ) and ClC5 ( F ) protein from Western blots shown in D . Untreated controls were incubated with serum-free medium containing 0.1% DMSO, the same concentration used in treated cells. For B, C, D, and F , all values were normalized against β-actin expression, and the control values were set to 100%. Values are mean ± SE of three experiments. * p

    Techniques Used: Western Blot, Incubation, Concentration Assay, Expressing

    3) Product Images from "Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts"

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts

    Journal:

    doi: 10.1158/0008-5472.CAN-07-5127

    Agents that disrupt the pH gradient of lysosomes enhance the matrix degradation and podosome formation. A).v-Src NIH3T3 cells cultured on cy3-gelatin-coated (red) coverslips were mock-treated (0.1%DMSO) or treated with 5 mM NH 4 Cl, 5 nM Bafilomycin A1,
    Figure Legend Snippet: Agents that disrupt the pH gradient of lysosomes enhance the matrix degradation and podosome formation. A).v-Src NIH3T3 cells cultured on cy3-gelatin-coated (red) coverslips were mock-treated (0.1%DMSO) or treated with 5 mM NH 4 Cl, 5 nM Bafilomycin A1,

    Techniques Used: Cell Culture

    4) Product Images from "Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism"

    Article Title: Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism

    Journal: Cancer & Metabolism

    doi: 10.1186/2049-3002-2-2

    Transfection of HCT-116 cells with PFKFB3 siRNA stimulates autophagy. LC3-II and p62 protein levels were determined using Western blotting 48 hours after transfection with either control (ctrl) or a siRNA directed against PFKFB3 (PFKFB3) (A) . Treatment with 1 nM bafilomycin A1 (Baf A1) was used to determine if LC3-II levels were a result of increased autophagic flux or impaired degradation (A) . Quantitative densitometry was performed to assess relative protein levels (B, C) . LC3-II and p62 levels are expressed as the mean fold change ± SD from three experiments relative to LC3-I or β-actin and control. After 48 hours of transfection with either control (ctrl) or PFKFB3-specific siRNA, cells also were stained with acridine orange, observed by fluorescent microscopy and collected by flow cytometry to measure the relative content of acidic compartments (D) . Examination of the cells by electron microscopy demonstrated that PFKFB3 siRNA transfection resulted in cells containing intracellular structures consistent with autophagosomes (E; arrow) . Data are presented as the mean ± SD from three experiments (* P
    Figure Legend Snippet: Transfection of HCT-116 cells with PFKFB3 siRNA stimulates autophagy. LC3-II and p62 protein levels were determined using Western blotting 48 hours after transfection with either control (ctrl) or a siRNA directed against PFKFB3 (PFKFB3) (A) . Treatment with 1 nM bafilomycin A1 (Baf A1) was used to determine if LC3-II levels were a result of increased autophagic flux or impaired degradation (A) . Quantitative densitometry was performed to assess relative protein levels (B, C) . LC3-II and p62 levels are expressed as the mean fold change ± SD from three experiments relative to LC3-I or β-actin and control. After 48 hours of transfection with either control (ctrl) or PFKFB3-specific siRNA, cells also were stained with acridine orange, observed by fluorescent microscopy and collected by flow cytometry to measure the relative content of acidic compartments (D) . Examination of the cells by electron microscopy demonstrated that PFKFB3 siRNA transfection resulted in cells containing intracellular structures consistent with autophagosomes (E; arrow) . Data are presented as the mean ± SD from three experiments (* P

    Techniques Used: Transfection, Western Blot, Staining, Microscopy, Flow Cytometry, Cytometry, Electron Microscopy

    PFKFB3 inhibition with 3PO stimulates autophagy. HCT-116 cells were treated with either vehicle, or 7.5, 10, or 15 μM 3PO for 24 hours and LC3-II and p62 expression was measured by Western blot (A) and densitometry (B, C) . Addition of bafilomycin A1 (Baf A1) was used to determine if the changes in LC3-II were the result of increased synthesis or impaired degradation. LC3-II quantitation is relative to control + bafilomycin due to the absence of a visible band in the control sample. HCT-116 cells were also stained with 1 μg/mL acridine orange for 15 minutes, viewed using a fluorescent microscope, harvested for flow cytometry and gating was used to quantitate the number of cells with a high AO fluorescence and expressed relative to vehicle (D, E) . Using electron microscopy, autophagic structures were seen in cells exposed to 3PO (F; arrow) .
    Figure Legend Snippet: PFKFB3 inhibition with 3PO stimulates autophagy. HCT-116 cells were treated with either vehicle, or 7.5, 10, or 15 μM 3PO for 24 hours and LC3-II and p62 expression was measured by Western blot (A) and densitometry (B, C) . Addition of bafilomycin A1 (Baf A1) was used to determine if the changes in LC3-II were the result of increased synthesis or impaired degradation. LC3-II quantitation is relative to control + bafilomycin due to the absence of a visible band in the control sample. HCT-116 cells were also stained with 1 μg/mL acridine orange for 15 minutes, viewed using a fluorescent microscope, harvested for flow cytometry and gating was used to quantitate the number of cells with a high AO fluorescence and expressed relative to vehicle (D, E) . Using electron microscopy, autophagic structures were seen in cells exposed to 3PO (F; arrow) .

    Techniques Used: Inhibition, Expressing, Western Blot, Quantitation Assay, Staining, Microscopy, Flow Cytometry, Cytometry, Fluorescence, Electron Microscopy

    5) Product Images from "A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications"

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001770

    Vacuole formation of HUVECs induced by βγ-CAT and inhibition by bafilomycin A1 and nocodazole. (A) Extensive cell vacuolation induced by high dosages of βγ-CAT. HUVECs planted on glass coverslips were incubated with βγ-CAT (25 nM) for 30 min. PBS-treated cells were used as a negative control and cells treated with NH 4 Cl (16 mM) for 18 h were used as a positive control. The cells were stained with neutral red as described in “ Methods ”. The vacuole formation in the cells was observed by confocal microscope. Scale bars equal to 10 µm. (B) Neutral red uptake of βγ-CAT treated HUVECs in the absence and presence of bafilomycin A1 and nocodazole. HUVECs (2.5×10 4 cells/ml) seeded in 96 well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C, then different concentrations of βγ-CAT (50 p M-5 nM) were added and incubated for another 30 min. Neutral red uptake was performed as described in “ Methods ”. (C) Inhibition of cell detachment induced by βγ-CAT in the presence of bafilomycin A1 and nocodazole. HUVECs planted in 24-well plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C, then βγ-CAT (1 nM, 5 nM and 10 nM) was added and incubated for 5 h. Cell detachment was analyzed as described in “ Methods ”. Data were expressed as means±SEM of triplicate measurements (#, p
    Figure Legend Snippet: Vacuole formation of HUVECs induced by βγ-CAT and inhibition by bafilomycin A1 and nocodazole. (A) Extensive cell vacuolation induced by high dosages of βγ-CAT. HUVECs planted on glass coverslips were incubated with βγ-CAT (25 nM) for 30 min. PBS-treated cells were used as a negative control and cells treated with NH 4 Cl (16 mM) for 18 h were used as a positive control. The cells were stained with neutral red as described in “ Methods ”. The vacuole formation in the cells was observed by confocal microscope. Scale bars equal to 10 µm. (B) Neutral red uptake of βγ-CAT treated HUVECs in the absence and presence of bafilomycin A1 and nocodazole. HUVECs (2.5×10 4 cells/ml) seeded in 96 well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C, then different concentrations of βγ-CAT (50 p M-5 nM) were added and incubated for another 30 min. Neutral red uptake was performed as described in “ Methods ”. (C) Inhibition of cell detachment induced by βγ-CAT in the presence of bafilomycin A1 and nocodazole. HUVECs planted in 24-well plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C, then βγ-CAT (1 nM, 5 nM and 10 nM) was added and incubated for 5 h. Cell detachment was analyzed as described in “ Methods ”. Data were expressed as means±SEM of triplicate measurements (#, p

    Techniques Used: Inhibition, Incubation, Negative Control, Positive Control, Staining, Microscopy

    Related Articles

    Clone Assay:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: For transfection of SP-C constructs cloned into pIRES2-EGFP, pEGFP-C1, or pcDNA3.1, HEK293 cells were seeded at 3–5 × 105 cells/well in a six-well plate. .. For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Positive Control:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: PBS-treated cells were used as a negative control and cells treated with NH4 Cl (16 mM) for 18 h were used as a positive control. .. To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Cytometry:

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production
    Article Snippet: Bafilomycin A1 experiments To establish the bafilomycin concentration to be used in subsequent virus rescue experiments, bafilomycin A1 (Sigma Aldrich; or DMSO vehicle control) was titrated onto mock-electroporated cells and both viability and intracellular pH assessed 24 hrs post-treatment. .. Cells were then washed with PBS, trypsinized, and LysoTracker Red content analyzed by flow cytometry after gating on live cell singlets.

    Construct:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: For transfection of SP-C constructs cloned into pIRES2-EGFP, pEGFP-C1, or pcDNA3.1, HEK293 cells were seeded at 3–5 × 105 cells/well in a six-well plate. .. For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Incubation:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: .. To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C. .. Then βγ-CAT (0–5 nM) was added and the cells were incubated for 30 min at 37°C.

    Article Title: Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17
    Article Snippet: For starvation treatment, cells were washed twice with PBS and incubated in amino acid–free DMEM (048-33575; Wako Pure Chemical Industries) without serum. .. For bafilomycin A1 , Torin 1, or wortmannin treatment, cells were cultured with 100 nM bafilomycin A1 (B1793; Sigma-Aldrich), 250 nM Torin 1 (4247; Tocris Bioscience), or 200 nM wortmannin (W1628; Sigma-Aldrich) for 2 h, respectively.

    Article Title: Cadmium Impairs Albumin Reabsorption by Down-regulating Megalin and ClC5 Channels in Renal Proximal Tubule Cells
    Article Snippet: CHX, bafilomycin A1, and MG-132 treatments Cycloheximide (CHX), bafilomycin A1 (Sigma), and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132) (Calbiochem, San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). .. LLC-PK1 cells were incubated with serum-free medium containing each chemical or DMSO alone at the same concentration used in treated cells (0.1% or 0.05%).

    Article Title: Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism
    Article Snippet: OptiMEM (Invitrogen) with 1% Lipofectamine RNAiMAX (Invitrogen) was incubated at RT for 5 minutes. siRNA was added to the Lipofectamine mixture and incubated for 20 minutes at room temperature. .. Samples in which bafilomycin A1 was used were treated with 1 nM bafilomycin A1 (Sigma, St. Louis, MO, USA) for 24 hours prior to harvest.

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: Effect of pH and bafilomycin A1 on the separation of Stx2a and CdtV-B from OMVs To investigate the effect of pH, 5791/99 OMVs (~10 μg of OMV protein) were incubated (1 h, 37°C) in 20 mM TRIS-HCl buffer with pH ranging from 8.0 to 5.0. .. To determine the effect of bafilomycin A1, HBMEC were pretreated with 100 nM bafilomycin A1 (Sigma-Aldrich) for 1 h at 37°C, and without removing the inhibitor, exposed (30 min at 4°C followed by 4 h at 37°C) to OMVs 5791/99.

    Activity Assay:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C. .. For testing the inhibitory activity of bafilomycin A1 and nocodazole on cellular functions of βγ-CAT, HUVECs in 24-well plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Cell Culture:

    Article Title: Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17
    Article Snippet: .. For bafilomycin A1 , Torin 1, or wortmannin treatment, cells were cultured with 100 nM bafilomycin A1 (B1793; Sigma-Aldrich), 250 nM Torin 1 (4247; Tocris Bioscience), or 200 nM wortmannin (W1628; Sigma-Aldrich) for 2 h, respectively. .. To visualize lysosomes, cells were cultured with 50 nM LysoTracker red DND-99 (L-7528; Thermo Fisher Scientific) for 1 h.

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: Paragraph title: Transfection and Cell Culture ... For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Transformation Assay:

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: The v-Src transformed NIH3T3 cells were plated on glass bottom chamber slides (BD Bioscience, San Jose, CA) and transfected with CFP-β-actin using the Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. .. For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA).

    Inhibition:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: Paragraph title: Measurement of cell vacuole formation and inhibition by bafilomycin A1 and nocodazole ... To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Transfection:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: Paragraph title: Transfection and Cell Culture ... For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Article Title: Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism
    Article Snippet: Paragraph title: siRNA transfection ... Samples in which bafilomycin A1 was used were treated with 1 nM bafilomycin A1 (Sigma, St. Louis, MO, USA) for 24 hours prior to harvest.

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: Twenty-four hours after transfection, cells were placed on the stage of a LSM 510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) and maintained at 37 °C in a CO2 -independent medium (Invitrogen, Eugene, OR) supplemented with 5% FCS. .. For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA).

    Infection:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: At 3 days after infection, mice were lavaged with saline and large aggregate (LA) surfactant isolated ( ); LBs were prepared from lung tissue homogenates, as described by Osanai and colleagues ( ). .. For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Confocal Laser Scanning Microscopy:

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: To determine the effect of bafilomycin A1, HBMEC were pretreated with 100 nM bafilomycin A1 (Sigma-Aldrich) for 1 h at 37°C, and without removing the inhibitor, exposed (30 min at 4°C followed by 4 h at 37°C) to OMVs 5791/99. .. The presence of Stx2a and CdtV-B in the endoplasmic reticulum was analyzed by CLSM as described above.

    Fluorescence:

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: Twenty-four hours after transfection, cells were placed on the stage of a LSM 510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) and maintained at 37 °C in a CO2 -independent medium (Invitrogen, Eugene, OR) supplemented with 5% FCS. .. For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA).

    Isolation:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: At 3 days after infection, mice were lavaged with saline and large aggregate (LA) surfactant isolated ( ); LBs were prepared from lung tissue homogenates, as described by Osanai and colleagues ( ). .. For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Flow Cytometry:

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production
    Article Snippet: Bafilomycin A1 experiments To establish the bafilomycin concentration to be used in subsequent virus rescue experiments, bafilomycin A1 (Sigma Aldrich; or DMSO vehicle control) was titrated onto mock-electroporated cells and both viability and intracellular pH assessed 24 hrs post-treatment. .. Cells were then washed with PBS, trypsinized, and LysoTracker Red content analyzed by flow cytometry after gating on live cell singlets.

    Microscopy:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: After washed with PBS again, the cells were observed immediately under a confocal microscope at 645 nm by 488 nm excitation. .. To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: Twenty-four hours after transfection, cells were placed on the stage of a LSM 510 fluorescence confocal microscope (Carl Zeiss, Thornwood, NY) and maintained at 37 °C in a CO2 -independent medium (Invitrogen, Eugene, OR) supplemented with 5% FCS. .. For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA).

    Mouse Assay:

    Article Title: Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
    Article Snippet: At 3 days after infection, mice were lavaged with saline and large aggregate (LA) surfactant isolated ( ); LBs were prepared from lung tissue homogenates, as described by Osanai and colleagues ( ). .. For bafilomycin A1 experiments, cells were treated 100 nM bafilomycin A1 (EMD Chemicals) for 4 hours, and harvested 24 hours later for analysis.

    Software:

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA). .. Data analysis was performed using LSM 510 software.

    Negative Control:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: PBS-treated cells were used as a negative control and cells treated with NH4 Cl (16 mM) for 18 h were used as a positive control. .. To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Article Title: Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism
    Article Snippet: siRNA transfection HCT-116 cells were plated at 100,000 cells/well in a 6-well dish in 2.5 mL complete medium and, 24 hours after seeding, were transfected with either control siRNA (Stealth Negative Control Medium GC Duplex) or PFKFB3 siRNA (HSS107860 or HSS107862) (all from Invitrogen, Grand Island, NY, USA). .. Samples in which bafilomycin A1 was used were treated with 1 nM bafilomycin A1 (Sigma, St. Louis, MO, USA) for 24 hours prior to harvest.

    Live Cell Imaging:

    Article Title: Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts
    Article Snippet: Paragraph title: Time-lapse live cell Imaging ... For Bafilomycin A1 experiments, cells were first loaded with lysosomal tracer for more than 15 minutes before adding 20 μM Bafilomycin A1 (Calbiochem, San Diego, CA).

    Concentration Assay:

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production
    Article Snippet: .. Bafilomycin A1 experiments To establish the bafilomycin concentration to be used in subsequent virus rescue experiments, bafilomycin A1 (Sigma Aldrich; or DMSO vehicle control) was titrated onto mock-electroporated cells and both viability and intracellular pH assessed 24 hrs post-treatment. .. Cellular viability was determined using CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer’s protocol.

    Article Title: Cadmium Impairs Albumin Reabsorption by Down-regulating Megalin and ClC5 Channels in Renal Proximal Tubule Cells
    Article Snippet: CHX, bafilomycin A1, and MG-132 treatments Cycloheximide (CHX), bafilomycin A1 (Sigma), and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG-132) (Calbiochem, San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). .. LLC-PK1 cells were incubated with serum-free medium containing each chemical or DMSO alone at the same concentration used in treated cells (0.1% or 0.05%).

    Article Title: Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism
    Article Snippet: The mixture was added to a single well of the 6-well plate for a total volume of 3 mL and a final siRNA concentration of 10 nM. .. Samples in which bafilomycin A1 was used were treated with 1 nM bafilomycin A1 (Sigma, St. Louis, MO, USA) for 24 hours prior to harvest.

    Staining:

    Article Title: A Novel Non-Lens ??-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications
    Article Snippet: Afterwards, the cells were washed with PBS and stained with 0.05% neutral red for 4 min at 37°C. .. To test the inhibitory effects of bafilomycin A1 and nocodazole (Sigma, USA) on cell vacuole formation induced by βγ-CAT, HUVECs in 96-well titer plates were incubated with bafilomycin A1 (25 nM) or nocodazole (20 µM) for 30 min at 37°C.

    Cell Viability Assay:

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production
    Article Snippet: Bafilomycin A1 experiments To establish the bafilomycin concentration to be used in subsequent virus rescue experiments, bafilomycin A1 (Sigma Aldrich; or DMSO vehicle control) was titrated onto mock-electroporated cells and both viability and intracellular pH assessed 24 hrs post-treatment. .. Cellular viability was determined using CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer’s protocol.

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  • 90
    Millipore baf
    Effect of <t>BAF</t> depletion on viral chromatin. ChIP assays were performed to determine the levels of histone H3, the H3K9me3 heterochromatin mark, and the H3K4me3 euchromatin mark on the <t>ICP27</t> IE gene promoter. (A to C) HeLa cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 at an MOI of 1 and fixed at the postinfection times indicated (top panels) or 2 hpi (bottom panels). Chromatin was prepared and immunoprecipitated with antibodies specific for H3 (A), H3K9me3 (B), and H3K4me3 (C), and the amounts of ICP27 promoter sequences were determined by quantitative PCR. The top panels present two independent experiments, and the bottom panels present three or more independent experiments. Lines connect values of individual experiments. (D and E) ChIP assays were performed to determine the levels of BAF (D) and SETD1A (E) on viral gene promoters. HA-BAF was expressed transiently in HeLa cells that were then infected with HSV-1 at an MOI of 5. FLAG-SETD1A was expressed transiently in BAF-depleted HeLa (siBAF) or control (NT) cells that were then infected with HSV-1 at an MOI of 5. At 2 hpi, the cells were fixed and ChIP was performed with antibodies specific for HA or FLAG. The levels of individual HA-BAF- or FLAG-SETD1A-associated promoter sequences were determined by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. *, P
    Baf, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baf/product/Millipore
    Average 90 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    baf - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    95
    Millipore bafilomycin a1
    Patient-derived SMC lines exhibit hyperactive mTORC1 signaling. (A) Simplified mTORC1 signaling network. (B) Representative western blot for pan-S6K and its T389 phosphorylated form (P-S6K) in SMC lines, and densitometry based quantification expressed as relative to control 120ls-SMCs. Average diameter of cells (C) and change in LC3B-II levels with or without <t>bafilomycin</t> A1 (D) in SMC lines. (E) Western blot for HIF1α (left panel) and densitometry-based quantification (right panel) expressed as relative to 121-SMC control. (F) Densitometry-based quantification of western blots for HIF1α expression in SMC lines in the presence of DMSO (vehicle) or rapamycin, expressed as relative to the DMSO condition for each cell line. Statistics are relative to 120ls-SMC (B, C, D), 121-SMC (E), and DMSO condition (F) (t-test). P values of
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bafilomycin a1/product/Millipore
    Average 95 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    bafilomycin a1 - by Bioz Stars, 2020-02
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    Effect of BAF depletion on viral chromatin. ChIP assays were performed to determine the levels of histone H3, the H3K9me3 heterochromatin mark, and the H3K4me3 euchromatin mark on the ICP27 IE gene promoter. (A to C) HeLa cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 at an MOI of 1 and fixed at the postinfection times indicated (top panels) or 2 hpi (bottom panels). Chromatin was prepared and immunoprecipitated with antibodies specific for H3 (A), H3K9me3 (B), and H3K4me3 (C), and the amounts of ICP27 promoter sequences were determined by quantitative PCR. The top panels present two independent experiments, and the bottom panels present three or more independent experiments. Lines connect values of individual experiments. (D and E) ChIP assays were performed to determine the levels of BAF (D) and SETD1A (E) on viral gene promoters. HA-BAF was expressed transiently in HeLa cells that were then infected with HSV-1 at an MOI of 5. FLAG-SETD1A was expressed transiently in BAF-depleted HeLa (siBAF) or control (NT) cells that were then infected with HSV-1 at an MOI of 5. At 2 hpi, the cells were fixed and ChIP was performed with antibodies specific for HA or FLAG. The levels of individual HA-BAF- or FLAG-SETD1A-associated promoter sequences were determined by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. *, P

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: Effect of BAF depletion on viral chromatin. ChIP assays were performed to determine the levels of histone H3, the H3K9me3 heterochromatin mark, and the H3K4me3 euchromatin mark on the ICP27 IE gene promoter. (A to C) HeLa cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 at an MOI of 1 and fixed at the postinfection times indicated (top panels) or 2 hpi (bottom panels). Chromatin was prepared and immunoprecipitated with antibodies specific for H3 (A), H3K9me3 (B), and H3K4me3 (C), and the amounts of ICP27 promoter sequences were determined by quantitative PCR. The top panels present two independent experiments, and the bottom panels present three or more independent experiments. Lines connect values of individual experiments. (D and E) ChIP assays were performed to determine the levels of BAF (D) and SETD1A (E) on viral gene promoters. HA-BAF was expressed transiently in HeLa cells that were then infected with HSV-1 at an MOI of 5. FLAG-SETD1A was expressed transiently in BAF-depleted HeLa (siBAF) or control (NT) cells that were then infected with HSV-1 at an MOI of 5. At 2 hpi, the cells were fixed and ChIP was performed with antibodies specific for HA or FLAG. The levels of individual HA-BAF- or FLAG-SETD1A-associated promoter sequences were determined by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. *, P

    Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

    Techniques: Chromatin Immunoprecipitation, Transfection, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Effect of BAF on viral transcript levels. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. (A) Knockdown efficiency was confirmed by quantitative RT-PCR. (B to D) Cells were harvested at the postinfection times indicated, total RNAs were prepared, and IE ( ICP4 [B] and ICP27 [C]) and E ( ICP8 [D]) gene mRNA levels were measured by quantitative RT-PCR. The mRNA levels were normalized to 18S rRNA. Results shown are means and standard deviations from three independent experiments.

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: Effect of BAF on viral transcript levels. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. (A) Knockdown efficiency was confirmed by quantitative RT-PCR. (B to D) Cells were harvested at the postinfection times indicated, total RNAs were prepared, and IE ( ICP4 [B] and ICP27 [C]) and E ( ICP8 [D]) gene mRNA levels were measured by quantitative RT-PCR. The mRNA levels were normalized to 18S rRNA. Results shown are means and standard deviations from three independent experiments.

    Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

    Techniques: Transfection, Infection, Quantitative RT-PCR

    BAF localizes to viral RCs. (A) HFF cells were infected with HSV-1 at an MOI of 10, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP8 at 8 hpi. Images were deconvoluted by the inverse filter algorithm in the AxioVision 4.8 image acquisition software. (B) HeLa cell expressing FLAG-BAF were infected with HSV-1 DG1 at an MOI of 50, harvested at 4 hpi, and immunoprecipitated (IP) with antibodies specific for FLAG or ICP8. ICP4, ICP8, VP16-GFP, and FLAG-BAF were detected with antibodies specific for ICP4, ICP8, GFP, and FLAG.

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: BAF localizes to viral RCs. (A) HFF cells were infected with HSV-1 at an MOI of 10, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP8 at 8 hpi. Images were deconvoluted by the inverse filter algorithm in the AxioVision 4.8 image acquisition software. (B) HeLa cell expressing FLAG-BAF were infected with HSV-1 DG1 at an MOI of 50, harvested at 4 hpi, and immunoprecipitated (IP) with antibodies specific for FLAG or ICP8. ICP4, ICP8, VP16-GFP, and FLAG-BAF were detected with antibodies specific for ICP4, ICP8, GFP, and FLAG.

    Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

    Techniques: Infection, Immunofluorescence, Software, Expressing, Immunoprecipitation

    Effect of BAF on nuclear entry of VP16, viral DNA, and localization to RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 DG1 at an MOI of 100. (A) The cells were harvested at 3 hpi and separated into cytoplasmic (Cyto) and nuclear (Nuc) fractions. VP16-GFP was detected with anti-GFP antibody (left). GAPDH and lamin B1 were used as loading controls. The relative levels of VP16-GFP in the cytoplasmic and nuclear fractions were quantified with Image Studio Lite (right). The histogram shows the mean values and standard deviations from two independent experiments. (B) HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. The cells were harvested at 2 hpi, nuclei were isolated, and vDNA levels were measured by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. (C) ChIP assays were performed to determine the levels of VP16-GFP on IE gene promoters. HFF cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 DG1 at an MOI of 5 and fixed at 3 hpi. ChIP was performed with an anti-GFP antibody, and the amounts of the ICP4 and ICP27 gene promoters immunoprecipitated were determined by quantitative PCR. Lines connect values of individual experiments. (D) HFF cells were transfected with NT control or BAF-specific siRNA, infected with HSV-1 at an MOI of 0.1, and fixed at 40 hpi, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP4. The nuclei were stained with DAPI. The histogram represents the mean values and standard deviations from three independent experiments ( n = > 50 per individual experiment).

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: Effect of BAF on nuclear entry of VP16, viral DNA, and localization to RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 DG1 at an MOI of 100. (A) The cells were harvested at 3 hpi and separated into cytoplasmic (Cyto) and nuclear (Nuc) fractions. VP16-GFP was detected with anti-GFP antibody (left). GAPDH and lamin B1 were used as loading controls. The relative levels of VP16-GFP in the cytoplasmic and nuclear fractions were quantified with Image Studio Lite (right). The histogram shows the mean values and standard deviations from two independent experiments. (B) HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. The cells were harvested at 2 hpi, nuclei were isolated, and vDNA levels were measured by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. (C) ChIP assays were performed to determine the levels of VP16-GFP on IE gene promoters. HFF cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 DG1 at an MOI of 5 and fixed at 3 hpi. ChIP was performed with an anti-GFP antibody, and the amounts of the ICP4 and ICP27 gene promoters immunoprecipitated were determined by quantitative PCR. Lines connect values of individual experiments. (D) HFF cells were transfected with NT control or BAF-specific siRNA, infected with HSV-1 at an MOI of 0.1, and fixed at 40 hpi, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP4. The nuclei were stained with DAPI. The histogram represents the mean values and standard deviations from three independent experiments ( n = > 50 per individual experiment).

    Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

    Techniques: Transfection, Infection, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation, Immunofluorescence, Staining

    Patient-derived SMC lines exhibit hyperactive mTORC1 signaling. (A) Simplified mTORC1 signaling network. (B) Representative western blot for pan-S6K and its T389 phosphorylated form (P-S6K) in SMC lines, and densitometry based quantification expressed as relative to control 120ls-SMCs. Average diameter of cells (C) and change in LC3B-II levels with or without bafilomycin A1 (D) in SMC lines. (E) Western blot for HIF1α (left panel) and densitometry-based quantification (right panel) expressed as relative to 121-SMC control. (F) Densitometry-based quantification of western blots for HIF1α expression in SMC lines in the presence of DMSO (vehicle) or rapamycin, expressed as relative to the DMSO condition for each cell line. Statistics are relative to 120ls-SMC (B, C, D), 121-SMC (E), and DMSO condition (F) (t-test). P values of

    Journal: Cancer research

    Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis

    doi: 10.1158/0008-5472.CAN-17-0925

    Figure Lengend Snippet: Patient-derived SMC lines exhibit hyperactive mTORC1 signaling. (A) Simplified mTORC1 signaling network. (B) Representative western blot for pan-S6K and its T389 phosphorylated form (P-S6K) in SMC lines, and densitometry based quantification expressed as relative to control 120ls-SMCs. Average diameter of cells (C) and change in LC3B-II levels with or without bafilomycin A1 (D) in SMC lines. (E) Western blot for HIF1α (left panel) and densitometry-based quantification (right panel) expressed as relative to 121-SMC control. (F) Densitometry-based quantification of western blots for HIF1α expression in SMC lines in the presence of DMSO (vehicle) or rapamycin, expressed as relative to the DMSO condition for each cell line. Statistics are relative to 120ls-SMC (B, C, D), 121-SMC (E), and DMSO condition (F) (t-test). P values of

    Article Snippet: For bafilomycin A1, torin-1, rapamycin experiments: cells were treated with 2 nM torin-1 (Millipore, #475991), 50 nM rapamycin (or DMSO for controls) for 4 hours, and then with 250 nM bafilomycin A1 (or DMSO for controls) (Enzo Life Sciences, # BML-CM110-0100) for an additional 2 hours.

    Techniques: Derivative Assay, Western Blot, Expressing, T-Test

    Selective toxicity of TSC-LAM patient SMCs by dual targeting of mTORC1 and autophagy signaling. (A) Representative western blots of LC3B-I and LC3B-II levels in control (120ls-SMCs) and P6-derived SMC lines to depict LC3B-II accumulation in vehicle versus +bafilomycin A1 conditions, and the corresponding response to mTOR inhibitors torin-1 and rapamycin. (B) Basal levels of LC3B-II, based on densitometry quantification of western blots and normalized to β-actin levels, in SMC lines under vehicle, torin-1 or rapamycin treatment. Statistics are relative to vehicle. (C) % of cells positive for propidium iodide (PI) relative to vehicle for each cell line, following a 24 hr treatment with 2.5 µM chloroquine (CQ), 20 nM rapamycin, or both. Measurements were collected via flow cytometry. P values of

    Journal: Cancer research

    Article Title: Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis

    doi: 10.1158/0008-5472.CAN-17-0925

    Figure Lengend Snippet: Selective toxicity of TSC-LAM patient SMCs by dual targeting of mTORC1 and autophagy signaling. (A) Representative western blots of LC3B-I and LC3B-II levels in control (120ls-SMCs) and P6-derived SMC lines to depict LC3B-II accumulation in vehicle versus +bafilomycin A1 conditions, and the corresponding response to mTOR inhibitors torin-1 and rapamycin. (B) Basal levels of LC3B-II, based on densitometry quantification of western blots and normalized to β-actin levels, in SMC lines under vehicle, torin-1 or rapamycin treatment. Statistics are relative to vehicle. (C) % of cells positive for propidium iodide (PI) relative to vehicle for each cell line, following a 24 hr treatment with 2.5 µM chloroquine (CQ), 20 nM rapamycin, or both. Measurements were collected via flow cytometry. P values of

    Article Snippet: For bafilomycin A1, torin-1, rapamycin experiments: cells were treated with 2 nM torin-1 (Millipore, #475991), 50 nM rapamycin (or DMSO for controls) for 4 hours, and then with 250 nM bafilomycin A1 (or DMSO for controls) (Enzo Life Sciences, # BML-CM110-0100) for an additional 2 hours.

    Techniques: Laser Capture Microdissection, Western Blot, Derivative Assay, Flow Cytometry, Cytometry

    Functional AT 1 Rs are localized to endolysosomes in hypothalamic neurons. A : ANG II-induced Ca 2+ response is abolished after 1 h of incubation with bafilomycin A1 (Baf, 1 μM) or rapamycin (Rap, 30 μM), but not brefeldin (Bref, 10 μM).

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Direct evidence of intracrine angiotensin II signaling in neurons

    doi: 10.1152/ajpcell.00131.2013

    Figure Lengend Snippet: Functional AT 1 Rs are localized to endolysosomes in hypothalamic neurons. A : ANG II-induced Ca 2+ response is abolished after 1 h of incubation with bafilomycin A1 (Baf, 1 μM) or rapamycin (Rap, 30 μM), but not brefeldin (Bref, 10 μM).

    Article Snippet: Bafilomycin A1, brefeldin A, ryanodine, and xestospongin C were obtained from EMD Millipore (Billerica, MA); ANG II, heparin, rapamycin, U-73122, SKF-96365, BAPTA-AM, 1-oleoyl-2-acetyl- sn -glycerol (OAG), and CV-11947 (candesartan) from Sigma-Aldrich (St. Louis, MO); and Ned-19 and PD-123319 ditrifluoroacetate from Tocris Bioscience (R & D Systems, Minneapolis, MN).

    Techniques: Functional Assay, Incubation

    SLC25A46 L341P is polyubiquitylated and degraded by the proteasome. (A) HEK293 T-REx Flp-In cells expressing 2xHA-SLC25A46 L341P were treated with MG132, bortezomib, bafilomycin A1, or DMSO (0.1%) for 6 h. Cells were lysed, and total extracts were analyzed by SDS–PAGE. Antibodies against LC3 and ubiquitin were included as controls to verify inhibition of autophagy and protein degradation, respectively. TOMM40 served as loading control. The asterisk marks monoubiquitylated 2xHA-SLC25A46 L341P. (B) SLC25A46 L341P accumulates on mitochondria upon proteasome inhibition. HEK293T cells were transfected as in A and then treated with 10 μM MG132 (M) or control DMSO (D) for 6 h. Mitochondria were isolated and lysed; HA-tagged SLC25A46 L341P was detected by immunoblotting. (C) Ubiquitylated SLC25A46 WT or L341P was immunoprecipitated under denaturing conditions with a Flag antibody from cells transiently expressing HA-ubiquitin-GFP and 3xFlag-SLC25A46 WT or L341P. Ubiquitin was detected with an anti-HA antibody. GFP cleaved from HA-ubiquitin served as transfection control, and TOMM40 was included as a loading control. (D) The ratio between ubiquitylated and nonubiquitylated SLC25A46 WT and L341P was determined after densitometric analysis of both proteins pools of C. Mean ± SEM n = 3. (E) Proteasome inhibition blocks the rapid degradation of SLC25A46 L341P in mitochondria. HEK293T were transiently transfected with constructs for 2xHA-SLC25A46 WT or L341P for 24 h and then harvested before (0) and 15 or 30 min after the addition of cycloheximide. Mitochondria were isolated and analyzed by SDS–PAGE and immunoblotting. To block proteasome-dependent degradation, 25 μM MG132 was added at the time as cycloheximide addition.

    Journal: Molecular Biology of the Cell

    Article Title: Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

    doi: 10.1091/mbc.E16-07-0545

    Figure Lengend Snippet: SLC25A46 L341P is polyubiquitylated and degraded by the proteasome. (A) HEK293 T-REx Flp-In cells expressing 2xHA-SLC25A46 L341P were treated with MG132, bortezomib, bafilomycin A1, or DMSO (0.1%) for 6 h. Cells were lysed, and total extracts were analyzed by SDS–PAGE. Antibodies against LC3 and ubiquitin were included as controls to verify inhibition of autophagy and protein degradation, respectively. TOMM40 served as loading control. The asterisk marks monoubiquitylated 2xHA-SLC25A46 L341P. (B) SLC25A46 L341P accumulates on mitochondria upon proteasome inhibition. HEK293T cells were transfected as in A and then treated with 10 μM MG132 (M) or control DMSO (D) for 6 h. Mitochondria were isolated and lysed; HA-tagged SLC25A46 L341P was detected by immunoblotting. (C) Ubiquitylated SLC25A46 WT or L341P was immunoprecipitated under denaturing conditions with a Flag antibody from cells transiently expressing HA-ubiquitin-GFP and 3xFlag-SLC25A46 WT or L341P. Ubiquitin was detected with an anti-HA antibody. GFP cleaved from HA-ubiquitin served as transfection control, and TOMM40 was included as a loading control. (D) The ratio between ubiquitylated and nonubiquitylated SLC25A46 WT and L341P was determined after densitometric analysis of both proteins pools of C. Mean ± SEM n = 3. (E) Proteasome inhibition blocks the rapid degradation of SLC25A46 L341P in mitochondria. HEK293T were transiently transfected with constructs for 2xHA-SLC25A46 WT or L341P for 24 h and then harvested before (0) and 15 or 30 min after the addition of cycloheximide. Mitochondria were isolated and analyzed by SDS–PAGE and immunoblotting. To block proteasome-dependent degradation, 25 μM MG132 was added at the time as cycloheximide addition.

    Article Snippet: Chemicals and antibodies MG132 (Enzo Biochem), NMS873 (Selleck Chemicals), bortezomib, bafilomycin A1, and MLN4924 (EMD Millipore) were dissolved in DMSO, stored at −80°C, and used at the indicated concentrations.

    Techniques: Expressing, SDS Page, Inhibition, Transfection, Isolation, Immunoprecipitation, Construct, Blocking Assay