nuclear factor κ b  (Millipore)


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    Structured Review

    Millipore nuclear factor κ b
    Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- <t>κ</t> B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of <t>SN50,</t> a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P
    Nuclear Factor κ B, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production"

    Article Title: Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production

    Journal: Journal of Immunology Research

    doi: 10.1155/2014/856154

    Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- κ B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of SN50, a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P
    Figure Legend Snippet: Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- κ B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of SN50, a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

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    other:

    Article Title: Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production
    Article Snippet: SN50, a nuclear factor-κ B (NF-κ B) inhibitor, was purchased from Calbiochem (La Jolla, CA).

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    Millipore nf κ b activation inhibitor ii jsh 23
    3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- <t>κ</t> B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor <t>JSH-23</t> blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is
    Nf κ B Activation Inhibitor Ii Jsh 23, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monensin
    TGF-β-dependent modulation of soluble mediators. Purified γδ T cells were initially activated by BrHPP and expanded in the presence of and IL-2 ± TGF-β and/or IL-15 ± TGF-β. (A) Four days after inital stimulation, the amount of different mediators in cell-free culture supernatants was quantified by a bead-based muliplex assay. Median values from 3 independent experiments are depicted. (B) After 15 days of expansion, the Vδ2 T cells were activated for 6 h with TPA and Ionomycin. <t>Monensin</t> (3 µM) was added 4 h before fixation and subsequent intracellular staining with the specific mAb for granzyme A (clone: CB9), granzyme B (clone: GB11), IFN-γ (clone: 4S.B3), IL-9 (clone: MH9A3), perforin (clone: dG9), TNF-α (clone: 359–81-11) and the appropriate isotype controls. The differential mean fluorescence intensities were Z-normalized within each experiment. Same symbols correspond to same donors, mean values of 5–11 experiments are represented by horizontal bars. (C) Vδ2 T cells were initially activated by BrHPP and expanded in the presence of IL-15 + TGF-β. After 15 days of expansion, Vδ2 T cells were restimulated with BrHPP or were left untreated and then were directly co-cultured with Panc89 cells (E/T, 12.5:1) in a RTCA assay. IgG1 (clone: 11711.11), anti-IL-9 (clone: MH9D1), anti-IFN-γ (clone: B27) were added to the co-coculture. The quotient of the specific lysis in co-cultures treated with specific mAb relative to the specific lysis in isotype control-treated co-cultures 4 h and 24 h after effector cell addition is depicted as mean values from 8 experiments. Asterisks indicate significant differences in the specific lysis between the antibody- and isotype-treated co-cultures. Error bars represent the SEM. Asterisks refer to significant differences according to Student´s t-test (*p ≤ 0.05; **p ≤ 0.01). E/T, effector to target ratio.
    Monensin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- κ B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor JSH-23 blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is

    Journal: Cell Death and Differentiation

    Article Title: Maximal adamantyl-substituted retinoid-related molecule-induced apoptosis requires NF-κB noncanonical and canonical pathway activation

    doi: 10.1038/cdd.2010.84

    Figure Lengend Snippet: 3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- κ B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor JSH-23 blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is

    Article Snippet: NF- κ B activation inhibitor II JSH-23, proteasome inhibitor MG132 and lysosomal inhibitor CA-074Me from EMD Biosciences (Gibbstown, NJ, USA); and Cdc37 shRNA expression vector from Open Biosystems (Frederick, MD, USA).

    Techniques: Activation Assay, Transduction, Labeling, Staining, Expressing, Binding Assay

    TGF-β-dependent modulation of soluble mediators. Purified γδ T cells were initially activated by BrHPP and expanded in the presence of and IL-2 ± TGF-β and/or IL-15 ± TGF-β. (A) Four days after inital stimulation, the amount of different mediators in cell-free culture supernatants was quantified by a bead-based muliplex assay. Median values from 3 independent experiments are depicted. (B) After 15 days of expansion, the Vδ2 T cells were activated for 6 h with TPA and Ionomycin. Monensin (3 µM) was added 4 h before fixation and subsequent intracellular staining with the specific mAb for granzyme A (clone: CB9), granzyme B (clone: GB11), IFN-γ (clone: 4S.B3), IL-9 (clone: MH9A3), perforin (clone: dG9), TNF-α (clone: 359–81-11) and the appropriate isotype controls. The differential mean fluorescence intensities were Z-normalized within each experiment. Same symbols correspond to same donors, mean values of 5–11 experiments are represented by horizontal bars. (C) Vδ2 T cells were initially activated by BrHPP and expanded in the presence of IL-15 + TGF-β. After 15 days of expansion, Vδ2 T cells were restimulated with BrHPP or were left untreated and then were directly co-cultured with Panc89 cells (E/T, 12.5:1) in a RTCA assay. IgG1 (clone: 11711.11), anti-IL-9 (clone: MH9D1), anti-IFN-γ (clone: B27) were added to the co-coculture. The quotient of the specific lysis in co-cultures treated with specific mAb relative to the specific lysis in isotype control-treated co-cultures 4 h and 24 h after effector cell addition is depicted as mean values from 8 experiments. Asterisks indicate significant differences in the specific lysis between the antibody- and isotype-treated co-cultures. Error bars represent the SEM. Asterisks refer to significant differences according to Student´s t-test (*p ≤ 0.05; **p ≤ 0.01). E/T, effector to target ratio.

    Journal: Oncoimmunology

    Article Title: TGF-β enhances the cytotoxic activity of Vδ2 T cells

    doi: 10.1080/2162402X.2018.1522471

    Figure Lengend Snippet: TGF-β-dependent modulation of soluble mediators. Purified γδ T cells were initially activated by BrHPP and expanded in the presence of and IL-2 ± TGF-β and/or IL-15 ± TGF-β. (A) Four days after inital stimulation, the amount of different mediators in cell-free culture supernatants was quantified by a bead-based muliplex assay. Median values from 3 independent experiments are depicted. (B) After 15 days of expansion, the Vδ2 T cells were activated for 6 h with TPA and Ionomycin. Monensin (3 µM) was added 4 h before fixation and subsequent intracellular staining with the specific mAb for granzyme A (clone: CB9), granzyme B (clone: GB11), IFN-γ (clone: 4S.B3), IL-9 (clone: MH9A3), perforin (clone: dG9), TNF-α (clone: 359–81-11) and the appropriate isotype controls. The differential mean fluorescence intensities were Z-normalized within each experiment. Same symbols correspond to same donors, mean values of 5–11 experiments are represented by horizontal bars. (C) Vδ2 T cells were initially activated by BrHPP and expanded in the presence of IL-15 + TGF-β. After 15 days of expansion, Vδ2 T cells were restimulated with BrHPP or were left untreated and then were directly co-cultured with Panc89 cells (E/T, 12.5:1) in a RTCA assay. IgG1 (clone: 11711.11), anti-IL-9 (clone: MH9D1), anti-IFN-γ (clone: B27) were added to the co-coculture. The quotient of the specific lysis in co-cultures treated with specific mAb relative to the specific lysis in isotype control-treated co-cultures 4 h and 24 h after effector cell addition is depicted as mean values from 8 experiments. Asterisks indicate significant differences in the specific lysis between the antibody- and isotype-treated co-cultures. Error bars represent the SEM. Asterisks refer to significant differences according to Student´s t-test (*p ≤ 0.05; **p ≤ 0.01). E/T, effector to target ratio.

    Article Snippet: For intracellular staining, cells were stimulated for 6 h with 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma Aldrich, Missouri, USA) and 1 µg/mL ionomycin (EMD Millipore/Calbiochem, Darmstadt, Germany) as indicated; 3 µM monensin (EMD Millipore/Calbiochem) was added 4 h before fixation in each case.

    Techniques: Purification, Staining, Fluorescence, Cell Culture, Lysis

    Treatment of cortical cultures with reagents that inhibit the synthesis and/or secretion of CSPGs leads to a retention of the Cat-315 CSPG in neurons with cell surface Cat-315 immunoreactivity. Primary cortical cultures from P0 rats were maintained for 7 d in serum-containing medium followed by an additional 8 hr without ( A , A ′) or with ( B , B ′) monensin and then fixed, permeabilized, and double labeled with Cat-315 and TuJ1. Cultures from E16 rats were maintained for 3 d in serum-containing medium and then maintained for an additional 10 d with ( D ) or without ( C ) β-xyloside. A , P0 cultures at 7 d plus 8 hr in control medium show robust Cat-315 surface-associated staining. A ′, The same culture visualized for TuJ1 shows that TuJ1-immunoreactive processes are Cat-315-positive. B , P0 cultures at 7 d plus 8 hr in monensin show a marked increase in intracellular Cat-315 immunoreactivity ( arrows ) and a reduction of Cat-315 labeling of neurites. B ′, TuJ1 immunroeactivity shows that TuJ1-labeled neurites lack surface-associated Cat-315 staining. C , Three-day-old E16 cultures maintained for an additional 10 d in control medium show Cat-315 labeling of neuron cell bodies, against a dense background of Cat-315-positive neurites. D , After 10 d of β-xyloside treatment, there is a marked decrease in Cat-315 labeling of neurites and a marked increase in intracellular Cat-315 immunoreactivity ( arrows ). These experiments provide evidence that the Cat-315 CSPG is synthesized by the neurons that carry cell surface Cat-315 immunoreactivity. Scale bars: A–D , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neurons Produce a Neuronal Cell Surface-Associated Chondroitin Sulfate Proteoglycan

    doi: 10.1523/JNEUROSCI.18-01-00174.1998

    Figure Lengend Snippet: Treatment of cortical cultures with reagents that inhibit the synthesis and/or secretion of CSPGs leads to a retention of the Cat-315 CSPG in neurons with cell surface Cat-315 immunoreactivity. Primary cortical cultures from P0 rats were maintained for 7 d in serum-containing medium followed by an additional 8 hr without ( A , A ′) or with ( B , B ′) monensin and then fixed, permeabilized, and double labeled with Cat-315 and TuJ1. Cultures from E16 rats were maintained for 3 d in serum-containing medium and then maintained for an additional 10 d with ( D ) or without ( C ) β-xyloside. A , P0 cultures at 7 d plus 8 hr in control medium show robust Cat-315 surface-associated staining. A ′, The same culture visualized for TuJ1 shows that TuJ1-immunoreactive processes are Cat-315-positive. B , P0 cultures at 7 d plus 8 hr in monensin show a marked increase in intracellular Cat-315 immunoreactivity ( arrows ) and a reduction of Cat-315 labeling of neurites. B ′, TuJ1 immunroeactivity shows that TuJ1-labeled neurites lack surface-associated Cat-315 staining. C , Three-day-old E16 cultures maintained for an additional 10 d in control medium show Cat-315 labeling of neuron cell bodies, against a dense background of Cat-315-positive neurites. D , After 10 d of β-xyloside treatment, there is a marked decrease in Cat-315 labeling of neurites and a marked increase in intracellular Cat-315 immunoreactivity ( arrows ). These experiments provide evidence that the Cat-315 CSPG is synthesized by the neurons that carry cell surface Cat-315 immunoreactivity. Scale bars: A–D , 50 μm.

    Article Snippet: For monensin (Calbiochem, La Jolla, CA), P0 cultures were maintained for 7 d and then incubated in monensin-containing medium (1 × 10−6 m ) for 8 additional hours.

    Techniques: Labeling, Staining, Synthesized

    LPC-induced ERK activation via muG2A is inhibited by monensin. (A) DO11.10 G2A siRNA cells as well as muG2A-RFP reconstituted cells were either untreated or pretreated with 10 μM monensin for 30 min at pH 7.2/37°C. Cells were spun and resuspended in fresh monensin medium containing either 10 μM LPC or 10 ng/ml SDF1-α. Aliquots of cells were taken at indicated time points and cell lysates were prepared. The level of phosphorylated ERK and total ERK were analyzed using Western blot. (B) Wild-type DO11.10 cells were treated and analyzed as in A.

    Journal: Molecular Biology of the Cell

    Article Title: Lysophosphatidylcholine-induced Surface Redistribution Regulates Signaling of the Murine G Protein-coupled Receptor G2A D⃞

    doi: 10.1091/mbc.E04-12-1044

    Figure Lengend Snippet: LPC-induced ERK activation via muG2A is inhibited by monensin. (A) DO11.10 G2A siRNA cells as well as muG2A-RFP reconstituted cells were either untreated or pretreated with 10 μM monensin for 30 min at pH 7.2/37°C. Cells were spun and resuspended in fresh monensin medium containing either 10 μM LPC or 10 ng/ml SDF1-α. Aliquots of cells were taken at indicated time points and cell lysates were prepared. The level of phosphorylated ERK and total ERK were analyzed using Western blot. (B) Wild-type DO11.10 cells were treated and analyzed as in A.

    Article Snippet: Monensin (Calbiochem, San Diego, CA) was dissolved in ethanol to obtain 50 mM stock and stored at –20°C.

    Techniques: Activation Assay, Western Blot

    The R-A mutation of the DRY motif (DAY muG2A) resulted in constitutive surface expression as well as monensin-insensitive signaling responses. Localization of DAY muG2A-GFP expressed in DO11.10 cells and Swiss3T3 cells were analyzed by microscopy (A) and by subcellular fractionation analysis (B) as described in text. (C) Similar ERK activation in response to 10 μM LPC at pH 7.2 was observed in DO11.10 G2A siRNA cells reconstituted with wt or DAY muG2A-RFP. (D) LPC-induced ERK activation is resistant to monensin treatment (10 μM, 30 min at pH 7.2/37°C) in DO11.10 G2A siRNA cells overexpressing the DAY muG2A mutant.

    Journal: Molecular Biology of the Cell

    Article Title: Lysophosphatidylcholine-induced Surface Redistribution Regulates Signaling of the Murine G Protein-coupled Receptor G2A D⃞

    doi: 10.1091/mbc.E04-12-1044

    Figure Lengend Snippet: The R-A mutation of the DRY motif (DAY muG2A) resulted in constitutive surface expression as well as monensin-insensitive signaling responses. Localization of DAY muG2A-GFP expressed in DO11.10 cells and Swiss3T3 cells were analyzed by microscopy (A) and by subcellular fractionation analysis (B) as described in text. (C) Similar ERK activation in response to 10 μM LPC at pH 7.2 was observed in DO11.10 G2A siRNA cells reconstituted with wt or DAY muG2A-RFP. (D) LPC-induced ERK activation is resistant to monensin treatment (10 μM, 30 min at pH 7.2/37°C) in DO11.10 G2A siRNA cells overexpressing the DAY muG2A mutant.

    Article Snippet: Monensin (Calbiochem, San Diego, CA) was dissolved in ethanol to obtain 50 mM stock and stored at –20°C.

    Techniques: Mutagenesis, Expressing, Microscopy, Fractionation, Activation Assay

    LPC-induced cell migration via muG2A is blocked by monensin. (A) DO11.10 G2A siRNA cells, as well as cells reconstituted with an siRNA-resistant form of muG2A fused to RFP, were either untreated or pretreated with 50 μM monensin for 1 h at pH 7.2/37°C. Cells (2 × 10 5 ) were added to the upper chamber of a 24-well plate. LPC (10 μM) or 20 ng/ml SDF1-α was added to the lower chamber as chemoattractant. Transmigrated cells were recovered after 2 h and counted. Data are presented as mean ± SE. (B) Wild-type DO11.10 cells were treated and analyzed as in A.

    Journal: Molecular Biology of the Cell

    Article Title: Lysophosphatidylcholine-induced Surface Redistribution Regulates Signaling of the Murine G Protein-coupled Receptor G2A D⃞

    doi: 10.1091/mbc.E04-12-1044

    Figure Lengend Snippet: LPC-induced cell migration via muG2A is blocked by monensin. (A) DO11.10 G2A siRNA cells, as well as cells reconstituted with an siRNA-resistant form of muG2A fused to RFP, were either untreated or pretreated with 50 μM monensin for 1 h at pH 7.2/37°C. Cells (2 × 10 5 ) were added to the upper chamber of a 24-well plate. LPC (10 μM) or 20 ng/ml SDF1-α was added to the lower chamber as chemoattractant. Transmigrated cells were recovered after 2 h and counted. Data are presented as mean ± SE. (B) Wild-type DO11.10 cells were treated and analyzed as in A.

    Article Snippet: Monensin (Calbiochem, San Diego, CA) was dissolved in ethanol to obtain 50 mM stock and stored at –20°C.

    Techniques: Migration

    Blockade of general recycling pathways via monensin inhibits LPC-mediated muG2A redistribution to the cell surface. DO11.10 cells overexpressing muG2A-GFP were either untreated or pretreated with 50 μM monensin for 1 h at 37°C in serum-free medium (pH 7.2). Viable cells were recovered using Ficoll gradient and treated with 10 μM LPC for 2 h before being analyzed by confocal microscopy (A) and subcellular fractionation (B). Representative images are shown. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Lysophosphatidylcholine-induced Surface Redistribution Regulates Signaling of the Murine G Protein-coupled Receptor G2A D⃞

    doi: 10.1091/mbc.E04-12-1044

    Figure Lengend Snippet: Blockade of general recycling pathways via monensin inhibits LPC-mediated muG2A redistribution to the cell surface. DO11.10 cells overexpressing muG2A-GFP were either untreated or pretreated with 50 μM monensin for 1 h at 37°C in serum-free medium (pH 7.2). Viable cells were recovered using Ficoll gradient and treated with 10 μM LPC for 2 h before being analyzed by confocal microscopy (A) and subcellular fractionation (B). Representative images are shown. Bar, 20 μm.

    Article Snippet: Monensin (Calbiochem, San Diego, CA) was dissolved in ethanol to obtain 50 mM stock and stored at –20°C.

    Techniques: Confocal Microscopy, Fractionation