Structured Review

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Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol <t>tetraacetic</t> acid <t>(EGTA)</t> for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p
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Images

1) Product Images from "Inflammatory Effects of Menthol vs. Non-menthol Cigarette Smoke Extract on Human Lung Epithelial Cells: A Double-Hit on TRPM8 by Reactive Oxygen Species and Menthol"

Article Title: Inflammatory Effects of Menthol vs. Non-menthol Cigarette Smoke Extract on Human Lung Epithelial Cells: A Double-Hit on TRPM8 by Reactive Oxygen Species and Menthol

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2017.00263

Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol tetraacetic acid (EGTA) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p
Figure Legend Snippet: Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol tetraacetic acid (EGTA) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p

Techniques Used: Fluorescence, Transfection, Plasmid Preparation

2) Product Images from "TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation"

Article Title: TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0143171

Rotundifolone-induced Ca 2+ transient in myocytes depend of extracellular Ca 2+ . (A) Representative recording of the Ca 2+ signals induced by rotundifolone (10 −3 and 3x10 -3 M) in freshly dispersed vascular smooth muscle cells from rat superior mesenteric artery. (B) Summary data showing the amplitude of the Ca 2+ signal induced after perfusion with different concentrations of rotundifolone (3x10 -4 , n = 9; 10 −3 , n = 5; 3x10 -3 M, n = 11). (C) Representative recording of Ca 2+ oscillations induced by rotundifolone (3x10 -3 M) in myocytes placed in Ca 2+ -free medium fortified with 2 mM ethylene glycol tetraacetic acid (EGTA). (D) Summary data demonstrating the decreased amplitude of the Ca 2+ signal induced by rotundifolone in Ca 2+ -free medium (n = 13). The Ca 2+ oscillations were measured and represented as increases in fluorescence intensity relative to baseline [DF (%) = (F-F0/F0)*100]. The data were examined using unpaired Student's t tests (rotundifolone with or without EGTA) and one-way ANOVA followed by the Bonferroni post-test. (** p
Figure Legend Snippet: Rotundifolone-induced Ca 2+ transient in myocytes depend of extracellular Ca 2+ . (A) Representative recording of the Ca 2+ signals induced by rotundifolone (10 −3 and 3x10 -3 M) in freshly dispersed vascular smooth muscle cells from rat superior mesenteric artery. (B) Summary data showing the amplitude of the Ca 2+ signal induced after perfusion with different concentrations of rotundifolone (3x10 -4 , n = 9; 10 −3 , n = 5; 3x10 -3 M, n = 11). (C) Representative recording of Ca 2+ oscillations induced by rotundifolone (3x10 -3 M) in myocytes placed in Ca 2+ -free medium fortified with 2 mM ethylene glycol tetraacetic acid (EGTA). (D) Summary data demonstrating the decreased amplitude of the Ca 2+ signal induced by rotundifolone in Ca 2+ -free medium (n = 13). The Ca 2+ oscillations were measured and represented as increases in fluorescence intensity relative to baseline [DF (%) = (F-F0/F0)*100]. The data were examined using unpaired Student's t tests (rotundifolone with or without EGTA) and one-way ANOVA followed by the Bonferroni post-test. (** p

Techniques Used: Fluorescence

Related Articles

Transfection:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Positron Emission Tomography:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Synthesized:

Article Title: Copolymers enhance selective bacterial community colonization for potential root zone applications
Article Snippet: .. Materials Polyacrylic acid (PAA) and the corresponding mannan grafted polyacrylic acid (PAA-manngraft ) were synthesized from anhydrous 99% acrylic acid containing monomethyl ether hydroquinone as inhibitor, 99% methylene bisacrylamide as crosslinker and ammonium persulphate as a water soluble initiator from Sigma Aldrich (Australia) according to Supporting Information S-1. .. The high molecular weight mannan, derived from Saccharomyces cerevisiae (Sigma Aldrich, M3640), is a linear chain of 1,4-linked β-D-mannopyranosyl residues having less than 5% of galactose , , was used without further purification.

Mutagenesis:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Construct:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Purification:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

SDS-Gel:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Concentration Assay:

Article Title: A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans
Article Snippet: .. For okadaic acid treatment, nematodes were placed on NGM plates containing 100 mM glucose, and then, okadaic acid (Sigma-Aldrich) was added to these plates to a final concentration of 30, 60, 120 and 240 nM and allowed to dry for approximately 20 minutes. ..

other:

Article Title: Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture
Article Snippet: ATP, histamine, NMDA, 5-hydroxytryptamine (5-HT), carbachol (CCh), and cyclopiazonic acid (CPA) were purchased from Sigma.

Expressing:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Western Blot:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

SDS Page:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Plasmid Preparation:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

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    2-APB and BAPTA-AM prevent viral-induced FAK phosphorylation. CaSki cells were serum starved overnight, and a synchronized infection with HSV-2(G) was conducted as described in Materials and methods. 100 μM 2-APB, 10 μM <t>nifedipine,</t> or control buffer (5% DMSO) was added to cells at the time of binding or penetration (temperature shift, 15 min, 37°C). Cells were treated with low pH citrate buffer, washed, and cell lysates were prepared 10 min after citrate treatment. Western blots were prepared, probed with anti-FAK pY 397 , and scanned by optical densitometry. Blots were then stripped and reprobed with mAb to total FAK. A representative blot is shown in A. Alternatively, cells were pretreated for 2 h with BAPTA-AM, <t>EGTA,</t> or 5% methanol in PBS (control), and then exposed to HSV-2(G). (B) Lysates were prepared 5 min after infection and analyzed by Western blotting for FAK phosphorylation. (C) Results obtained from three independent experiments are summarized graphically as odu of phosphorylated FAK:odu total FAK as a percentage of control (HSV-2(G) in the absence of any inhibitors). Results are the mean ± SD obtained from three independent experiments; asterisks indicate P
    Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
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    BMTs activate AMPK through CaMKK and not through intracellular Ca 2+ changes. Serum-starved L6 myotubes were treated with either vehicle (V) alone, <t>BMT-17</t> at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot ( A ) and quantified by densitometry ( B ). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM <t>EGTA-AM</t> (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK in vitro kinase assay ( C ). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p
    Egta Am, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    2-APB and BAPTA-AM prevent viral-induced FAK phosphorylation. CaSki cells were serum starved overnight, and a synchronized infection with HSV-2(G) was conducted as described in Materials and methods. 100 μM 2-APB, 10 μM nifedipine, or control buffer (5% DMSO) was added to cells at the time of binding or penetration (temperature shift, 15 min, 37°C). Cells were treated with low pH citrate buffer, washed, and cell lysates were prepared 10 min after citrate treatment. Western blots were prepared, probed with anti-FAK pY 397 , and scanned by optical densitometry. Blots were then stripped and reprobed with mAb to total FAK. A representative blot is shown in A. Alternatively, cells were pretreated for 2 h with BAPTA-AM, EGTA, or 5% methanol in PBS (control), and then exposed to HSV-2(G). (B) Lysates were prepared 5 min after infection and analyzed by Western blotting for FAK phosphorylation. (C) Results obtained from three independent experiments are summarized graphically as odu of phosphorylated FAK:odu total FAK as a percentage of control (HSV-2(G) in the absence of any inhibitors). Results are the mean ± SD obtained from three independent experiments; asterisks indicate P

    Journal: The Journal of Cell Biology

    Article Title: Herpes simplex virus triggers activation of calcium-signaling pathways

    doi: 10.1083/jcb.200301084

    Figure Lengend Snippet: 2-APB and BAPTA-AM prevent viral-induced FAK phosphorylation. CaSki cells were serum starved overnight, and a synchronized infection with HSV-2(G) was conducted as described in Materials and methods. 100 μM 2-APB, 10 μM nifedipine, or control buffer (5% DMSO) was added to cells at the time of binding or penetration (temperature shift, 15 min, 37°C). Cells were treated with low pH citrate buffer, washed, and cell lysates were prepared 10 min after citrate treatment. Western blots were prepared, probed with anti-FAK pY 397 , and scanned by optical densitometry. Blots were then stripped and reprobed with mAb to total FAK. A representative blot is shown in A. Alternatively, cells were pretreated for 2 h with BAPTA-AM, EGTA, or 5% methanol in PBS (control), and then exposed to HSV-2(G). (B) Lysates were prepared 5 min after infection and analyzed by Western blotting for FAK phosphorylation. (C) Results obtained from three independent experiments are summarized graphically as odu of phosphorylated FAK:odu total FAK as a percentage of control (HSV-2(G) in the absence of any inhibitors). Results are the mean ± SD obtained from three independent experiments; asterisks indicate P

    Article Snippet: Reagents Verapamil, nifedipine, 2-APB, Tg, EGTA, EGTA-AM, and ionomycin were purchased from Calbiochem and diluted in DMSO or PBS per manufacturer's instructions.

    Techniques: Infection, Binding Assay, Western Blot

    The ER Ca 2+ release is required for HSV infection. The effects of pretreating cells for 1 h with 50 μM BAPTA-AM, 0.5 mM EGTA, or adding 100 μM 2-APB, 10 μM nifedipine, or 10 μM verapamil during viral penetration on HSV-1(KOS), HSV-2(G), or VSV infection of Vero cells (top) or CaSki cells (bottom) were examined. Results are presented as pfu formed in the presence of the indicated concentration of drug as a percentage of pfu formed in cells treated with control buffer (5% DMSO for pharmacological inhibitors or 5% methanol for BAPTA), and are means of at least three independent experiments conducted in duplicate. Cytotoxicity of pharmacological agents was determined in parallel after a 2-h exposure to drug and quantifying viable, proliferating cells at 24 h by MTS assay. Cell viability results are expressed as odu reading in the presence of the drug as a percentage of odu reading in the presence of control buffer, and are means of two independent experiments conducted in triplicate. Asterisks denote P

    Journal: The Journal of Cell Biology

    Article Title: Herpes simplex virus triggers activation of calcium-signaling pathways

    doi: 10.1083/jcb.200301084

    Figure Lengend Snippet: The ER Ca 2+ release is required for HSV infection. The effects of pretreating cells for 1 h with 50 μM BAPTA-AM, 0.5 mM EGTA, or adding 100 μM 2-APB, 10 μM nifedipine, or 10 μM verapamil during viral penetration on HSV-1(KOS), HSV-2(G), or VSV infection of Vero cells (top) or CaSki cells (bottom) were examined. Results are presented as pfu formed in the presence of the indicated concentration of drug as a percentage of pfu formed in cells treated with control buffer (5% DMSO for pharmacological inhibitors or 5% methanol for BAPTA), and are means of at least three independent experiments conducted in duplicate. Cytotoxicity of pharmacological agents was determined in parallel after a 2-h exposure to drug and quantifying viable, proliferating cells at 24 h by MTS assay. Cell viability results are expressed as odu reading in the presence of the drug as a percentage of odu reading in the presence of control buffer, and are means of two independent experiments conducted in triplicate. Asterisks denote P

    Article Snippet: Reagents Verapamil, nifedipine, 2-APB, Tg, EGTA, EGTA-AM, and ionomycin were purchased from Calbiochem and diluted in DMSO or PBS per manufacturer's instructions.

    Techniques: Infection, Concentration Assay, MTS Assay

    Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Functional cleavage of the common cytokine receptor ? chain (?c) by calpain

    doi:

    Figure Lengend Snippet: Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

    Article Snippet: Lysates were centrifuged at 15,000 rpm for 20 min at 4°C and incubated with CaCl2 (final concentration 5 mM) or EGTA (final concentration 10 mM) at 37°C for 0 to 60 min. To determine the specificity of proteolysis by calpain, calpastatin (20 μM, Calbiochem), an endogenous protease inhibitor that acts specifically on calpain, or antipain [S-(1 carboxy-2-phenylethyl)- l -carbamyl= l -arginyl- l -valylargininal] (Calbiochem), another potent inhibitor of calpain, were incubated in the presence of 5 mM CaCl2 .

    Techniques: In Vitro, Sequencing, Incubation, Protease Inhibitor, Immunoprecipitation, Staining

    BMTs activate AMPK through CaMKK and not through intracellular Ca 2+ changes. Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot ( A ) and quantified by densitometry ( B ). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK in vitro kinase assay ( C ). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p

    Journal: PLoS ONE

    Article Title: Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKK?

    doi: 10.1371/journal.pone.0062309

    Figure Lengend Snippet: BMTs activate AMPK through CaMKK and not through intracellular Ca 2+ changes. Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot ( A ) and quantified by densitometry ( B ). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK in vitro kinase assay ( C ). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p

    Article Snippet: The inability of EGTA-AM to block the BMT-dependent AMPK activation demonstrated that the BMT-dependent AMPK activation was independent of intracellular calcium levels.

    Techniques: Lysis, Western Blot, Isolation, Immunoprecipitation, In Vitro, Kinase Assay

    Dynamin I dephosphorylation is arrested by BAPTA ‐ AM and EGTA ‐ AM . Cultures were preincubated with BAPTA ‐ AM , EGTA ‐ AM (both 100 μM) or DMSO (vehicle control) for 30 min. They were then either stimulated with a train of 800 action potentials (80 Hz) or left to rest for 10 s. Cultures were then immediately lysed in SDS sample buffer. (a) Representative immunoblots of cerebellar granule neuron lysates probed with either dynamin I phospho‐Ser774 ( PD ynI) or synaptophysin (Syp) antibodies. (b) Quantification of PD ynI levels after normalisation against Syp ( PD ynI/Syp) ± SEM . Blue bars represent Ctrl, red bars BAPTA ‐ AM and purple bars EGTA ‐ AM , *** p

    Journal: Journal of Neurochemistry

    Article Title: Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity‐dependent bulk endocytosis

    doi: 10.1111/jnc.13132

    Figure Lengend Snippet: Dynamin I dephosphorylation is arrested by BAPTA ‐ AM and EGTA ‐ AM . Cultures were preincubated with BAPTA ‐ AM , EGTA ‐ AM (both 100 μM) or DMSO (vehicle control) for 30 min. They were then either stimulated with a train of 800 action potentials (80 Hz) or left to rest for 10 s. Cultures were then immediately lysed in SDS sample buffer. (a) Representative immunoblots of cerebellar granule neuron lysates probed with either dynamin I phospho‐Ser774 ( PD ynI) or synaptophysin (Syp) antibodies. (b) Quantification of PD ynI levels after normalisation against Syp ( PD ynI/Syp) ± SEM . Blue bars represent Ctrl, red bars BAPTA ‐ AM and purple bars EGTA ‐ AM , *** p

    Article Snippet: The inhibition of dynamin I dephosphorylation in the presence of EGTA‐AM is consistent with this event being central in the triggering of this endocytosis mode.

    Techniques: De-Phosphorylation Assay, Western Blot