Structured Review

Tocris ionomycin
High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with <t>ionomycin</t> (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P
Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ionomycin/product/Tocris
Average 93 stars, based on 14 article reviews
Price from $9.99 to $1999.99
ionomycin - by Bioz Stars, 2020-09
93/100 stars

Images

1) Product Images from "Lysosomal Ca2+ Signaling Regulates High Glucose-Mediated Interleukin-1β Secretion via Transcription Factor EB in Human Monocytic Cells"

Article Title: Lysosomal Ca2+ Signaling Regulates High Glucose-Mediated Interleukin-1β Secretion via Transcription Factor EB in Human Monocytic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01161

High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with ionomycin (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P
Figure Legend Snippet: High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with ionomycin (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P

Techniques Used: Expressing, Translocation Assay, Western Blot, Immunofluorescence

2) Product Images from "mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile"

Article Title: mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

Journal: PLoS ONE

doi: 10.1371/journal.pone.0154564

AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P
Figure Legend Snippet: AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

Techniques Used: In Vitro, Isolation, Mouse Assay, Labeling, Staining, Expressing

3) Product Images from "Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells"

Article Title: Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072881

Time course of the effects of [Ca 2+ ] i -elevating stimuli on ERK1/2 phosphorylation in salivary gland cell lines. HSY cells (A) and Par-C10 cells (B) were exposed to TG (1 µM) and ionomycin (1 µM) for various times, as indicated. Values shown are for ERK1/2 phosphorylations (normalized to total ERK2) relative to basal conditions.
Figure Legend Snippet: Time course of the effects of [Ca 2+ ] i -elevating stimuli on ERK1/2 phosphorylation in salivary gland cell lines. HSY cells (A) and Par-C10 cells (B) were exposed to TG (1 µM) and ionomycin (1 µM) for various times, as indicated. Values shown are for ERK1/2 phosphorylations (normalized to total ERK2) relative to basal conditions.

Techniques Used:

Time course of the effects of [Ca 2+ ] i -elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells. A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE 2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p
Figure Legend Snippet: Time course of the effects of [Ca 2+ ] i -elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells. A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE 2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p

Techniques Used:

4) Product Images from "ADAM10 sheddase activation is controlled by cell membrane asymmetry"

Article Title: ADAM10 sheddase activation is controlled by cell membrane asymmetry

Journal: Journal of Molecular Cell Biology

doi: 10.1093/jmcb/mjz008

Overexpression of ANO6 enhances ADAM10 sheddase activity upon calcium influx. ( A ) Mock-transfected and Anoctamin-6 (ANO6)-GFP-transfected COS7 cells were stimulated with ionomycin (IO, 1 μM) for the indicated time. After stimulation, cells were stained with Annexin V-APC and analysed via FACS analysis. ( B and C ) COS7 cells were co-transfected with ANO6-GFP or mock vector and the AP-tagged ADAM10 substrates BTC or VE-cadherin, respectively. Cells were stimulated with IO (1 μM) for 30 min. IO-induced shedding was significantly increased upon overexpression of ANO6. TAPI (10 μM), ADAM10 inhibitor GI (3 μM), and OPS (10 mM), but not OPC (10 mM), significantly abrogated the induced shedding. * indicates a significant increase compared to mock-transfected stimulated cells; # indicates significant decrease compared to ANO6-transfected stimulated control cells ( P
Figure Legend Snippet: Overexpression of ANO6 enhances ADAM10 sheddase activity upon calcium influx. ( A ) Mock-transfected and Anoctamin-6 (ANO6)-GFP-transfected COS7 cells were stimulated with ionomycin (IO, 1 μM) for the indicated time. After stimulation, cells were stained with Annexin V-APC and analysed via FACS analysis. ( B and C ) COS7 cells were co-transfected with ANO6-GFP or mock vector and the AP-tagged ADAM10 substrates BTC or VE-cadherin, respectively. Cells were stimulated with IO (1 μM) for 30 min. IO-induced shedding was significantly increased upon overexpression of ANO6. TAPI (10 μM), ADAM10 inhibitor GI (3 μM), and OPS (10 mM), but not OPC (10 mM), significantly abrogated the induced shedding. * indicates a significant increase compared to mock-transfected stimulated cells; # indicates significant decrease compared to ANO6-transfected stimulated control cells ( P

Techniques Used: Over Expression, Activity Assay, Transfection, Staining, FACS, Plasmid Preparation

PS interaction is required for ADAM10 activation. ( A and B ) COS7 cells were transfected with the AP-tagged ADAM10 substrate BTC and stimulated with ionomycin (IO, 1 μM; A ) or melittin (Mel, 0.5 μM; B ) for 30 min. Shedding was dose-dependently reduced by addition of the competing phosphatidylserine head group (OPS) but not by the head group of phosphatidylcholine (OPC). OPS (10 mM), broadspectrum metalloprotease inhibitor TAPI-1 (10 μM), and ADAM10 inhibitor GI (3 μM) significantly abrogated the induced shedding. ( C ) COS7 cells were transfected with the AP-tagged ADAM10 substrate VE-cadherin and stimulated with ionomycin for 30 min in the presence of OPS (10 mM), OPC (10 mM), TAPI-1 (10 μM), or ADAM10 inhibitor GI (3 μM) and analysed for substrate shedding. ( D ) Shedding of full-length (FL) E-cadherin was monitored by immunoblot analysis. HaCaT keratinocytes were stimulated with ionomycin (1 μM) in the presence of TAPI-1 (10 μM), OPS (10 mM), OPC (10 mM), or ADAM10 inhibitor GI (3 μM). ( E ) Densitometric quantification of E-cadherin C-terminal fragment (CTF) generation of three independent western blots. * indicates a significant increase compared to unstimulated cells; # indicates significant decrease compared to stimulated control ( P
Figure Legend Snippet: PS interaction is required for ADAM10 activation. ( A and B ) COS7 cells were transfected with the AP-tagged ADAM10 substrate BTC and stimulated with ionomycin (IO, 1 μM; A ) or melittin (Mel, 0.5 μM; B ) for 30 min. Shedding was dose-dependently reduced by addition of the competing phosphatidylserine head group (OPS) but not by the head group of phosphatidylcholine (OPC). OPS (10 mM), broadspectrum metalloprotease inhibitor TAPI-1 (10 μM), and ADAM10 inhibitor GI (3 μM) significantly abrogated the induced shedding. ( C ) COS7 cells were transfected with the AP-tagged ADAM10 substrate VE-cadherin and stimulated with ionomycin for 30 min in the presence of OPS (10 mM), OPC (10 mM), TAPI-1 (10 μM), or ADAM10 inhibitor GI (3 μM) and analysed for substrate shedding. ( D ) Shedding of full-length (FL) E-cadherin was monitored by immunoblot analysis. HaCaT keratinocytes were stimulated with ionomycin (1 μM) in the presence of TAPI-1 (10 μM), OPS (10 mM), OPC (10 mM), or ADAM10 inhibitor GI (3 μM). ( E ) Densitometric quantification of E-cadherin C-terminal fragment (CTF) generation of three independent western blots. * indicates a significant increase compared to unstimulated cells; # indicates significant decrease compared to stimulated control ( P

Techniques Used: Activation Assay, Transfection, Western Blot

Deletion of cationic amino acids in the ADAM10 stalk region impairs sheddase function. ( A ) A potential PS-binding motif (R657/K659/K660) was exchanged creating an ADAM10 stalk mutant (ADAM10-stalk Mut). ( B ) ADAM17/ADAM10 double-deficient HEK cells were co-transfected with BTC-AP and WT-ADAM10 (A10-WT), inactive ADAM10 (A10 E/A), or ADAM10 mutant and stimulated with ionomycin (IO, 1 μM) or melittin (Mel, 1 μM) for 30 min. AP-activity in the supernatant was calculated in relation to total (supernatant and cell pellet) AP activity and is shown in comparison to A10-WT as ‘control’. * indicates a significant increase compared to unstimulated cells; # indicates significant decrease compared to A10-WT transfected stimulated cells ( P
Figure Legend Snippet: Deletion of cationic amino acids in the ADAM10 stalk region impairs sheddase function. ( A ) A potential PS-binding motif (R657/K659/K660) was exchanged creating an ADAM10 stalk mutant (ADAM10-stalk Mut). ( B ) ADAM17/ADAM10 double-deficient HEK cells were co-transfected with BTC-AP and WT-ADAM10 (A10-WT), inactive ADAM10 (A10 E/A), or ADAM10 mutant and stimulated with ionomycin (IO, 1 μM) or melittin (Mel, 1 μM) for 30 min. AP-activity in the supernatant was calculated in relation to total (supernatant and cell pellet) AP activity and is shown in comparison to A10-WT as ‘control’. * indicates a significant increase compared to unstimulated cells; # indicates significant decrease compared to A10-WT transfected stimulated cells ( P

Techniques Used: Binding Assay, Mutagenesis, Transfection, Activity Assay

5) Product Images from "Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype"

Article Title: Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140050

CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members. (A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (E rev ) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean E rev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p
Figure Legend Snippet: CLCA4b does not evoke a calcium-dependent chloride current characteristic of other CLCA family members. (A) Currents were evoked in HEK293 cells expressing CLCA4b (wt) by a series of voltage steps (150 ms, -110 to -10 mV) before (left) and after (right) application of the calcium ionophore ionomycin (10 μM). Ionomycin stimulated an inward current at the holding potential of -50 mV and increased the amplitude of step-evoked currents. (B) Similar experiment for HEK293 cells expressing CLCA4b (mut). In A and B, zero current is indicated by the dotted lines. (C) Current-voltage relationship for the difference currents were obtained by subtracting the step-evoked currents before from those after ionomycin application. Linear extrapolation was used to determine the reversal potential (E rev ) for the ionomycin-stimulated currents which were normalized to the current at the holding potential (-50 mV). (D) Mean E rev for ionomycin-stimulated currents in cells expressing CLCA4b (wt) (n = 10), CLCA4b (mut) (n = 13), CLCA1 (n = 8), serving as a positive control, or EGFP alone (n = 8), which served as a negative control. *p

Techniques Used: Expressing, Mass Spectrometry, Positive Control, Negative Control

6) Product Images from "Identification of select G-protein coupled receptors as regulators of the ER-mitochondria contact by drug screening"

Article Title: Identification of select G-protein coupled receptors as regulators of the ER-mitochondria contact by drug screening

Journal: bioRxiv

doi: 10.1101/2020.05.11.088815

Elevated cytosolic Ca 2+ level enhances ER-Mito contact. A , Split-Rluc activity in DMSO, histamine, ionomycin, or CPA treated cells (n = 9 wells, DMSO, histamine, and CPA; n = 6, ionomycin; **p = 0.0017, *p = 0.0256, **** p
Figure Legend Snippet: Elevated cytosolic Ca 2+ level enhances ER-Mito contact. A , Split-Rluc activity in DMSO, histamine, ionomycin, or CPA treated cells (n = 9 wells, DMSO, histamine, and CPA; n = 6, ionomycin; **p = 0.0017, *p = 0.0256, **** p

Techniques Used: Activity Assay

7) Product Images from "Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines"

Article Title: Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

Journal: Cell Cycle

doi: 10.4161/15384101.2014.947759

( A ) Correlation of STAT4 and STAT6 expression with different T helper phenotype markers in CTCL cell lines under normal control vs. T cell stimulation culturing conditions (e.g., 10 ng/mL of PMA (phorbol 12-myristate 13-acetate) and 1 (M of ionomycin
Figure Legend Snippet: ( A ) Correlation of STAT4 and STAT6 expression with different T helper phenotype markers in CTCL cell lines under normal control vs. T cell stimulation culturing conditions (e.g., 10 ng/mL of PMA (phorbol 12-myristate 13-acetate) and 1 (M of ionomycin

Techniques Used: Expressing, Cell Stimulation

8) Product Images from "PKCα integrates spatiotemporally distinct Ca2+ and autocrine BDNF signaling to facilitate synaptic plasticity"

Article Title: PKCα integrates spatiotemporally distinct Ca2+ and autocrine BDNF signaling to facilitate synaptic plasticity

Journal: Nature neuroscience

doi: 10.1038/s41593-018-0184-3

Development of FLIM-FRET sensors for isozyme-specific PKC activity A) ITRACK sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: mCh is targeted to membrane with K-CAAX domain. B) IDOCKS sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: The pseudosubstrate domain from PKCα/β is tagged by 2mCh fluorophores. ) before and after PKC activation by PdBu (1 μM) and ionomycin (1 μM). Warmer colors indicate a decrease in lifetime and an increase in PKCα activity (Quantification in (E)). Scale bar = 20 μm. D) Same as in C but cells express IDOCKSα or IDOCKSα neg (Quantification in (E)). E) Mean time course of the change in fluorescence lifetime of PKCα sensors upon stimulation with PdBU (1 μM) and ionomycin (1 μM) or vehicle (n [experiments/cells]: Veh=6/30, ITRACKα= 7/37, ITRACKα neg= 5/28, IDOCKSα= 6/29, IDOCKSα neg= 6/36, shaded regions indicate SEM). F) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα (shown in E), PKCβ and PKCγ in HeLa cells as indicated (n [experiments/cells]: ITRACKβ= 6/29, IDOCKSβ=7/44, ITRACKγ= 6/30, IDOCKSγ= 6/27, Bars indicate mean and SEM, symbols indicate individual cells). G) Representative fluorescent lifetime images of secondary apical dendrites of CA1 hippocampal neurons expressing ITRACKα or IDOCKSα before or 2 mins after stimulation with PdBu (1 μM) and NMDA (20 μM). Scale bar = 2 μm. H) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα, PKCβ, and PKCγ in neurons as indicated (n [experiments/neurons] =5/5 for all conditions, Bars indicate mean and SEM, symbols indicate individual neurons).
Figure Legend Snippet: Development of FLIM-FRET sensors for isozyme-specific PKC activity A) ITRACK sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: mCh is targeted to membrane with K-CAAX domain. B) IDOCKS sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: The pseudosubstrate domain from PKCα/β is tagged by 2mCh fluorophores. ) before and after PKC activation by PdBu (1 μM) and ionomycin (1 μM). Warmer colors indicate a decrease in lifetime and an increase in PKCα activity (Quantification in (E)). Scale bar = 20 μm. D) Same as in C but cells express IDOCKSα or IDOCKSα neg (Quantification in (E)). E) Mean time course of the change in fluorescence lifetime of PKCα sensors upon stimulation with PdBU (1 μM) and ionomycin (1 μM) or vehicle (n [experiments/cells]: Veh=6/30, ITRACKα= 7/37, ITRACKα neg= 5/28, IDOCKSα= 6/29, IDOCKSα neg= 6/36, shaded regions indicate SEM). F) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα (shown in E), PKCβ and PKCγ in HeLa cells as indicated (n [experiments/cells]: ITRACKβ= 6/29, IDOCKSβ=7/44, ITRACKγ= 6/30, IDOCKSγ= 6/27, Bars indicate mean and SEM, symbols indicate individual cells). G) Representative fluorescent lifetime images of secondary apical dendrites of CA1 hippocampal neurons expressing ITRACKα or IDOCKSα before or 2 mins after stimulation with PdBu (1 μM) and NMDA (20 μM). Scale bar = 2 μm. H) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα, PKCβ, and PKCγ in neurons as indicated (n [experiments/neurons] =5/5 for all conditions, Bars indicate mean and SEM, symbols indicate individual neurons).

Techniques Used: Activity Assay, Activation Assay, Fluorescence, Expressing

9) Product Images from "Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling"

Article Title: Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087014

5-HT 4d receptor-stimulated APP shedding requires Src and phospholipase C, but not PKC or calcium. (A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT 4d , were treated with 1 µM prucalopride or 5-HT (5-HT 4 receptor agonists) and PMA in the absence or presence of 50 µM Bosutinib (Src inhibitor) (A), 30 µM D609 (PLC inhibitor) (B) or 2 µM GF109203X (PKC inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. Values shown are mean ± SEM of 6 individual wells and were normalized towards vehicle control. (D) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT 4d , were loaded with Fluo-4 NW mix and fluorescence of the calcium-sensitive dye in each well was recorded at the baseline (F 0 ) and after stimulation with 1 µM prucalopride, 30 µM ATP (purinergic ionotropic receptors agonist), 20 µM Ionomycin (calcium ionophore) or DMSO (F). Calcium response shown is a ratio of maximum fluorescence intensity at 40 sec to baseline fluorescence (F/F 0 ). Values shown are mean ± SEM of 2 individual wells and were normalized to vehicle control. * P
Figure Legend Snippet: 5-HT 4d receptor-stimulated APP shedding requires Src and phospholipase C, but not PKC or calcium. (A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT 4d , were treated with 1 µM prucalopride or 5-HT (5-HT 4 receptor agonists) and PMA in the absence or presence of 50 µM Bosutinib (Src inhibitor) (A), 30 µM D609 (PLC inhibitor) (B) or 2 µM GF109203X (PKC inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. Values shown are mean ± SEM of 6 individual wells and were normalized towards vehicle control. (D) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT 4d , were loaded with Fluo-4 NW mix and fluorescence of the calcium-sensitive dye in each well was recorded at the baseline (F 0 ) and after stimulation with 1 µM prucalopride, 30 µM ATP (purinergic ionotropic receptors agonist), 20 µM Ionomycin (calcium ionophore) or DMSO (F). Calcium response shown is a ratio of maximum fluorescence intensity at 40 sec to baseline fluorescence (F/F 0 ). Values shown are mean ± SEM of 2 individual wells and were normalized to vehicle control. * P

Techniques Used: Transfection, Planar Chromatography, Fluorescence, Size-exclusion Chromatography

10) Product Images from "Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6) *"

Article Title: Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.534156

Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p
Figure Legend Snippet: Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p

Techniques Used: Transfection, Construct, Confocal Microscopy

11) Product Images from "The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function"

Article Title: The Alzheimer’s disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function

Journal: bioRxiv

doi: 10.1101/2020.04.27.059600

Receptor-mediated release of Ca 2+ in macrophages and microglia. A . Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2 P522 mice (P522, blue) and Plcg2 R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. B . Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2 R522 mice (blue: P522) and Plcg2 R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data in A and B shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests. PLCG2 R522 hiPSC-derived microglia show increased cytosolic Ca 2+ influx in comparison to controls following activation of PLCγ2 using anti-CD32. Cytoplasmic Ca2+ increase following activation of PLCγ2 using anti-CD32 Representative Ca 2+ traces ( C ) and graphical summary ( D ). 3 independent PLCG2 R522 CRISPR-engineered clones were examined. Data shown is mean±SD of 4 independent experiments and were analysed by one-way ANOVA with Tukey’s multiple comparison test (** = p
Figure Legend Snippet: Receptor-mediated release of Ca 2+ in macrophages and microglia. A . Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2 P522 mice (P522, blue) and Plcg2 R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. B . Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2 R522 mice (blue: P522) and Plcg2 R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data in A and B shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests. PLCG2 R522 hiPSC-derived microglia show increased cytosolic Ca 2+ influx in comparison to controls following activation of PLCγ2 using anti-CD32. Cytoplasmic Ca2+ increase following activation of PLCγ2 using anti-CD32 Representative Ca 2+ traces ( C ) and graphical summary ( D ). 3 independent PLCG2 R522 CRISPR-engineered clones were examined. Data shown is mean±SD of 4 independent experiments and were analysed by one-way ANOVA with Tukey’s multiple comparison test (** = p

Techniques Used: Derivative Assay, Mouse Assay, Generated, Activation Assay, CRISPR, Clone Assay

Specificity of PLCγ2 in response of macrophages/microglia to physiologically-relevant stimuli. A . M-MØP (blue: P522; red: R522) were loaded with Fluo-8 Ca 2+ indicator and examined for peak changes in fluoresce after exposure to 5ug/ml anti-FcγRII/III (2.4G2) with or without pre-exposure for 2 hours with Edelfosine (10µM) or U73122 (5µM) or Xestospogin C (5µM). B . Left panel shows LNA GapmeR percentage knockdown of Plcg2 gene expression in M-MOP cells within the P522 control (blue) and R522 (red) variant lines. Right panel shows Fura2 340/380 time traces from M-MØP cell lines (blue: P522; red: R522) which have undergone Plcg2 knockdown (square symbols) using an antisense LNA GapmeR. Cells were exposed to 5µg/ml anti-FcγRII/III and 2µM Ionomycin. C . Microglia from Plcg2 P522 (blue: P522) mice and Plcg2 R522 mice (red: R522) from neonate cortex (solid colour) or hippocampus (striped) were loaded with Fluo-8 Ca 2+ indicator. These cells were then examined for peak changes in fluoresce after exposure to 5µg/ml anti-FcγRII/III. These readings were taken with or without pre-exposure for 2 hours with Edelfosine (10µM) or U73122 (5µM). D Cortical microglia from Plcg2 P522 (blue: P522) mice and Plcg2 R522 mice (red: R522) from neonate cortex (solid colour) or hippocampus (striped) were loaded with Fluo-8 Ca 2+ indicator. These cells were then examined for peak changes in fluoresce after exposure to LPS (50ng/ml) or Aβ 1-42 oligomers (40µM). Data shown as the mean±SD of 3 independent experiments. Data in A, C and D were analysed by two-way ANOVA with Dunnett’s multiple Comparison test. See also Supplementary figure 3.
Figure Legend Snippet: Specificity of PLCγ2 in response of macrophages/microglia to physiologically-relevant stimuli. A . M-MØP (blue: P522; red: R522) were loaded with Fluo-8 Ca 2+ indicator and examined for peak changes in fluoresce after exposure to 5ug/ml anti-FcγRII/III (2.4G2) with or without pre-exposure for 2 hours with Edelfosine (10µM) or U73122 (5µM) or Xestospogin C (5µM). B . Left panel shows LNA GapmeR percentage knockdown of Plcg2 gene expression in M-MOP cells within the P522 control (blue) and R522 (red) variant lines. Right panel shows Fura2 340/380 time traces from M-MØP cell lines (blue: P522; red: R522) which have undergone Plcg2 knockdown (square symbols) using an antisense LNA GapmeR. Cells were exposed to 5µg/ml anti-FcγRII/III and 2µM Ionomycin. C . Microglia from Plcg2 P522 (blue: P522) mice and Plcg2 R522 mice (red: R522) from neonate cortex (solid colour) or hippocampus (striped) were loaded with Fluo-8 Ca 2+ indicator. These cells were then examined for peak changes in fluoresce after exposure to 5µg/ml anti-FcγRII/III. These readings were taken with or without pre-exposure for 2 hours with Edelfosine (10µM) or U73122 (5µM). D Cortical microglia from Plcg2 P522 (blue: P522) mice and Plcg2 R522 mice (red: R522) from neonate cortex (solid colour) or hippocampus (striped) were loaded with Fluo-8 Ca 2+ indicator. These cells were then examined for peak changes in fluoresce after exposure to LPS (50ng/ml) or Aβ 1-42 oligomers (40µM). Data shown as the mean±SD of 3 independent experiments. Data in A, C and D were analysed by two-way ANOVA with Dunnett’s multiple Comparison test. See also Supplementary figure 3.

Techniques Used: Expressing, Variant Assay, Mouse Assay

12) Product Images from "TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis"

Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0127480

Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].
Figure Legend Snippet: Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Microscopy, Transfection

Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .
Figure Legend Snippet: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.
Figure Legend Snippet: TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.

Techniques Used: Over Expression, Expressing, Marker, Fluorescence, Western Blot, Flow Cytometry, Cytometry

13) Product Images from "Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane"

Article Title: Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

Journal: mBio

doi: 10.1128/mBio.01552-15

EHV-1 triggers the increase of cytosolic Ca 2+ . (A) ED cells were loaded with Fura-2AM, and live fluorescent images were taken every 5 s prior to and following the addition of EHV-1, ionomycin, EHV-4, or Ca 2+ -free medium (MEM) at time point 50 s. Shown is one representative image captured at each of the indicated time points. (B) The curves shown refer to the average of three independent experiments of fluorescence intensities of Fura-2AM versus time of excited ED cells being exposed to EHV-1, EHV-4, ionomycin, or MEM. P
Figure Legend Snippet: EHV-1 triggers the increase of cytosolic Ca 2+ . (A) ED cells were loaded with Fura-2AM, and live fluorescent images were taken every 5 s prior to and following the addition of EHV-1, ionomycin, EHV-4, or Ca 2+ -free medium (MEM) at time point 50 s. Shown is one representative image captured at each of the indicated time points. (B) The curves shown refer to the average of three independent experiments of fluorescence intensities of Fura-2AM versus time of excited ED cells being exposed to EHV-1, EHV-4, ionomycin, or MEM. P

Techniques Used: Fluorescence

14) Product Images from "The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells"

Article Title: The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047825

KCa3.1 channels are absent in adult’s healthy brain and cerebellum, but are highly expressed in glioblastoma tumor samples and derived primary cell lines. ( A ) Real-time PCR on three primary human cell lines of glioblastomas (CRL8, lane 1; FCN9, lane 2; MZC12, lane 3) demonstrated that KCa3.1 transcripts expression is higher compared to NHA (lane 4). ( B–G ) Immunohistochemical staining on normal human brain tissue revealed KCa3.1 protein presence only in endothelial cells of blood vessels ( B ) while we observed a diffuse staining in high grade tumors (CRL8, C ; FCN9, D , MZC12, E ) and a KCa3.1 signal only in neo-vascularisation area (glio low grade, F). As positive control we used physiological lung tissue ( G ). ( H ) Typical time course of the current recorded at −40 mV from a FCN9 cell by applying repetitive (every 5s) voltage ramps from −100 to +100 mV. 3 mM TEA and 1 mM octanole were added to block the BK and gap junctional channel, respectively, usually co-expressed with KCa3.1 channels in glioblastoma cells (21; 22). DC-EBIO (100 µM)+ ionomycin (0.5 µM) and DC-EBIO+ion +3 µM TRAM-34 were applied in succession to verify the functional expression of KCa3.1 currents (cf text). ( I ) Representative I–V relationships in presence of DC-EBIO+ionomycin, and DC-EBIO+ ionomycin +TRAM-34. Data in panel ( H ) and ( I ) are from the same experiment. Inset : I-V relationship of the KCa3.1 current obtained by subtracting the current ramps recorded in DC-EBIO+ion+TRAM-34 from that recorded in DC-EBIO+ion. ( L ) Plot reporting the KCa3.1 current density at 0 mV (assessed as in panel I ) measured in the three primary glioblastoma cell lines. Since in several cells a voltage-gated K current activating at membrane potentials higher than −20 mV was present, in these cases measurements of the KCa3.1 current density were performed at −40 mV, and the assessed current density was then extrapolated at 0 mV by assuming a linear current-voltage relationship. *ANOVA test, p
Figure Legend Snippet: KCa3.1 channels are absent in adult’s healthy brain and cerebellum, but are highly expressed in glioblastoma tumor samples and derived primary cell lines. ( A ) Real-time PCR on three primary human cell lines of glioblastomas (CRL8, lane 1; FCN9, lane 2; MZC12, lane 3) demonstrated that KCa3.1 transcripts expression is higher compared to NHA (lane 4). ( B–G ) Immunohistochemical staining on normal human brain tissue revealed KCa3.1 protein presence only in endothelial cells of blood vessels ( B ) while we observed a diffuse staining in high grade tumors (CRL8, C ; FCN9, D , MZC12, E ) and a KCa3.1 signal only in neo-vascularisation area (glio low grade, F). As positive control we used physiological lung tissue ( G ). ( H ) Typical time course of the current recorded at −40 mV from a FCN9 cell by applying repetitive (every 5s) voltage ramps from −100 to +100 mV. 3 mM TEA and 1 mM octanole were added to block the BK and gap junctional channel, respectively, usually co-expressed with KCa3.1 channels in glioblastoma cells (21; 22). DC-EBIO (100 µM)+ ionomycin (0.5 µM) and DC-EBIO+ion +3 µM TRAM-34 were applied in succession to verify the functional expression of KCa3.1 currents (cf text). ( I ) Representative I–V relationships in presence of DC-EBIO+ionomycin, and DC-EBIO+ ionomycin +TRAM-34. Data in panel ( H ) and ( I ) are from the same experiment. Inset : I-V relationship of the KCa3.1 current obtained by subtracting the current ramps recorded in DC-EBIO+ion+TRAM-34 from that recorded in DC-EBIO+ion. ( L ) Plot reporting the KCa3.1 current density at 0 mV (assessed as in panel I ) measured in the three primary glioblastoma cell lines. Since in several cells a voltage-gated K current activating at membrane potentials higher than −20 mV was present, in these cases measurements of the KCa3.1 current density were performed at −40 mV, and the assessed current density was then extrapolated at 0 mV by assuming a linear current-voltage relationship. *ANOVA test, p

Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Positive Control, Blocking Assay, Functional Assay

Functional KCa3.1 channels are expressed in U87MG and GL261 cell lines. ( A ) Immunoblot analysis of U87MG and GL261 showed an expression of KCa3.1 channels correlated to transcript levels. ( B ) ( C ) Cytofluorimetric analysis of KCa3.1 on U87MG, GL261 and mouse normal adult astrocytes (NMA). Cells were incubated with anti-KCa3.1 followed by AlexaFluor488-conjugated Goat anti-Rabbit antibody as reported in the Materials and methods section. Ten thousand events were recorded and analyzed with Cyflogic. Gray histograms: cellular autofluorescence; black histograms: AlexaFluor488-conjugated Goat anti-Rabbit alone; green histograms: anti-KCa3.1 ( D ) Typical time course of the KCa3.1 current from a GL261 cell, recorded from I-V curves at 0 mV, in control conditions, after application of DC-EBIO (100 µM) + ionomycin (500 nM), and following application of 3 µM TRAM-34 in the continuous presence of DC-EBIO+ionomycin. Voltage ramps were applied every 5 s. Filled circles are data points obtained immediately before the I-V curves shown in panel E. ( E ) Representative I-V curves obtained by applying voltage ramps from −100 to +50 mV from a holding potential of 0 mV, in control conditions (CTRL), following the application of DC-EBIO+ionomycin, and after addition of TRAM-34 in the continuous presence of DC-EBIO+ionomycin. ( F ) Mean KCa3.1 current density measured in mouse NMA, as control, in GL261 and U87MG glioblastoma cell lines at 0 mV, assessed as the difference between the peak current density in DC-EBIO+ionomycin and the residual current following the addition of TRAM-34 (cf. filled circles in panel D ).
Figure Legend Snippet: Functional KCa3.1 channels are expressed in U87MG and GL261 cell lines. ( A ) Immunoblot analysis of U87MG and GL261 showed an expression of KCa3.1 channels correlated to transcript levels. ( B ) ( C ) Cytofluorimetric analysis of KCa3.1 on U87MG, GL261 and mouse normal adult astrocytes (NMA). Cells were incubated with anti-KCa3.1 followed by AlexaFluor488-conjugated Goat anti-Rabbit antibody as reported in the Materials and methods section. Ten thousand events were recorded and analyzed with Cyflogic. Gray histograms: cellular autofluorescence; black histograms: AlexaFluor488-conjugated Goat anti-Rabbit alone; green histograms: anti-KCa3.1 ( D ) Typical time course of the KCa3.1 current from a GL261 cell, recorded from I-V curves at 0 mV, in control conditions, after application of DC-EBIO (100 µM) + ionomycin (500 nM), and following application of 3 µM TRAM-34 in the continuous presence of DC-EBIO+ionomycin. Voltage ramps were applied every 5 s. Filled circles are data points obtained immediately before the I-V curves shown in panel E. ( E ) Representative I-V curves obtained by applying voltage ramps from −100 to +50 mV from a holding potential of 0 mV, in control conditions (CTRL), following the application of DC-EBIO+ionomycin, and after addition of TRAM-34 in the continuous presence of DC-EBIO+ionomycin. ( F ) Mean KCa3.1 current density measured in mouse NMA, as control, in GL261 and U87MG glioblastoma cell lines at 0 mV, assessed as the difference between the peak current density in DC-EBIO+ionomycin and the residual current following the addition of TRAM-34 (cf. filled circles in panel D ).

Techniques Used: Functional Assay, Expressing, Incubation

15) Product Images from "PKCα integrates spatiotemporally distinct Ca2+ and autocrine BDNF signaling to facilitate synaptic plasticity"

Article Title: PKCα integrates spatiotemporally distinct Ca2+ and autocrine BDNF signaling to facilitate synaptic plasticity

Journal: Nature neuroscience

doi: 10.1038/s41593-018-0184-3

Development of FLIM-FRET sensors for isozyme-specific PKC activity A) ITRACK sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: mCh is targeted to membrane with K-CAAX domain. B) IDOCKS sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: The pseudosubstrate domain from PKCα/β is tagged by 2mCh fluorophores. C) Representative fluorescent lifetime images of HeLa cells expressing ITRACKα or ITRACKα neg (see Fig. S1A ) before and after PKC activation by PdBu (1 μM) and ionomycin (1 μM). Warmer colors indicate a decrease in lifetime and an increase in PKCα activity (Quantification in (E)). Scale bar = 20 μm. D) Same as in C but cells express IDOCKSα or IDOCKSα neg (Quantification in (E)). E) Mean time course of the change in fluorescence lifetime of PKCα sensors upon stimulation with PdBU (1 μM) and ionomycin (1 μM) or vehicle (n [experiments/cells]: Veh=6/30, ITRACKα= 7/37, ITRACKα neg= 5/28, IDOCKSα= 6/29, IDOCKSα neg= 6/36, shaded regions indicate SEM). F) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα (shown in E), PKCβ and PKCγ in HeLa cells as indicated (n [experiments/cells]: ITRACKβ= 6/29, IDOCKSβ=7/44, ITRACKγ= 6/30, IDOCKSγ= 6/27, Bars indicate mean and SEM, symbols indicate individual cells). G) Representative fluorescent lifetime images of secondary apical dendrites of CA1 hippocampal neurons expressing ITRACKα or IDOCKSα before or 2 mins after stimulation with PdBu (1 μM) and NMDA (20 μM). Scale bar = 2 μm. H) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα, PKCβ, and PKCγ in neurons as indicated (n [experiments/neurons] =5/5 for all conditions, Bars indicate mean and SEM, symbols indicate individual neurons).
Figure Legend Snippet: Development of FLIM-FRET sensors for isozyme-specific PKC activity A) ITRACK sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: mCh is targeted to membrane with K-CAAX domain. B) IDOCKS sensor design. Donor: Classic PKC isozyme is tagged with mEGFP. Acceptor: The pseudosubstrate domain from PKCα/β is tagged by 2mCh fluorophores. C) Representative fluorescent lifetime images of HeLa cells expressing ITRACKα or ITRACKα neg (see Fig. S1A ) before and after PKC activation by PdBu (1 μM) and ionomycin (1 μM). Warmer colors indicate a decrease in lifetime and an increase in PKCα activity (Quantification in (E)). Scale bar = 20 μm. D) Same as in C but cells express IDOCKSα or IDOCKSα neg (Quantification in (E)). E) Mean time course of the change in fluorescence lifetime of PKCα sensors upon stimulation with PdBU (1 μM) and ionomycin (1 μM) or vehicle (n [experiments/cells]: Veh=6/30, ITRACKα= 7/37, ITRACKα neg= 5/28, IDOCKSα= 6/29, IDOCKSα neg= 6/36, shaded regions indicate SEM). F) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα (shown in E), PKCβ and PKCγ in HeLa cells as indicated (n [experiments/cells]: ITRACKβ= 6/29, IDOCKSβ=7/44, ITRACKγ= 6/30, IDOCKSγ= 6/27, Bars indicate mean and SEM, symbols indicate individual cells). G) Representative fluorescent lifetime images of secondary apical dendrites of CA1 hippocampal neurons expressing ITRACKα or IDOCKSα before or 2 mins after stimulation with PdBu (1 μM) and NMDA (20 μM). Scale bar = 2 μm. H) Quantification of drug-induced fluorescence lifetime changes of ITRACK and IDOCKS for PKCα, PKCβ, and PKCγ in neurons as indicated (n [experiments/neurons] =5/5 for all conditions, Bars indicate mean and SEM, symbols indicate individual neurons).

Techniques Used: Activity Assay, Expressing, Activation Assay, Fluorescence

16) Product Images from "Pathogenic Roles of Ion Channels and Transporters: Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl− secretion in murine colon by downregulating TMEM16A"

Article Title: Pathogenic Roles of Ion Channels and Transporters: Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl− secretion in murine colon by downregulating TMEM16A

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00328.2017

Effect of ionomycin on carbachol (CCH)-induced changes in short-circuit current ( I SC ) from control and acute dextran sulfate sodium (DSS)-colitis mice. A : representative trace of measured I SC following basolateral 100 μM CCH administration to control mucosa. B : representative trace of I SC from DSS-colitis mucosa following basolateral 100 μM CCH administration. C : group data of Δ I SC following CCH-administration in control and DSS mice. D : representative trace of I SC from control mucosa that was stimulated with 3 μM ionomycin (IONO) and followed by 100 μM CCH. E : group data of Δ I SC with 3 μM IONO followed by 100 μM CCH in control mucosa. F : group data of CCH-induced Δ I SC with and without 3 μM IONO administration. G : representative trace of I SC from mucosa exposed to DSS administered 3 μM IONO and 100 μM CCH. H : group data of Δ I SC from DSS-colitis in response to 3 μM IONO followed by 100 μM CCH administration. I : group data of DSS-colitis CCH-induced Δ I SC with and without 3 μM IONO administration. All presented bar graphs are summarized data that represent means ± SE. * P
Figure Legend Snippet: Effect of ionomycin on carbachol (CCH)-induced changes in short-circuit current ( I SC ) from control and acute dextran sulfate sodium (DSS)-colitis mice. A : representative trace of measured I SC following basolateral 100 μM CCH administration to control mucosa. B : representative trace of I SC from DSS-colitis mucosa following basolateral 100 μM CCH administration. C : group data of Δ I SC following CCH-administration in control and DSS mice. D : representative trace of I SC from control mucosa that was stimulated with 3 μM ionomycin (IONO) and followed by 100 μM CCH. E : group data of Δ I SC with 3 μM IONO followed by 100 μM CCH in control mucosa. F : group data of CCH-induced Δ I SC with and without 3 μM IONO administration. G : representative trace of I SC from mucosa exposed to DSS administered 3 μM IONO and 100 μM CCH. H : group data of Δ I SC from DSS-colitis in response to 3 μM IONO followed by 100 μM CCH administration. I : group data of DSS-colitis CCH-induced Δ I SC with and without 3 μM IONO administration. All presented bar graphs are summarized data that represent means ± SE. * P

Techniques Used: Mouse Assay

17) Product Images from "Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium"

Article Title: Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Journal: eLife

doi: 10.7554/eLife.57907

Activity measurements in HEK-TM cells. ( A ) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of IBMX to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.
Figure Legend Snippet: Activity measurements in HEK-TM cells. ( A ) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM). bPAC-mCherry was co-expressed with the mNphp3(201)-tagged mCherry nanobody. Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). bPAC activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression with the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). bPAC activity was determined according to the maximum amplitude of the Ca 2+ signal after light stimulation (465 nm light pulse, 1 s, 162 µW/cm²) compared to the ionomycin-evoked Ca 2+ signal. 5 min before light stimulation, cells were treated with 25 µM of IBMX to inhibit phosphodiesterases and sustain a long-lasting increase in cAMP. NT: non-transfected cells. ( B ) LAPD activity measurements in HEK-TM cells. To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylate cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). LAPD activity was determined in the presence of mNphp3(201)-VHH LaM-2 or mNphp3(201)-VHH LaM-4 (fused to HA or eGFP). Co-expression of the ciliary protein Sstr3-eGFP was used as a negative control (ciliary localized protein, but not binding to bPAC or LAPD). Fluo4-AM-loaded HEK-TM cells were incubated with 100 μM NKH477 during continuous 850 nm light illumination (0.5 µW/cm²). When reaching a steady-state, light was switched to 690 nm (0.5 µW/cm²) to stimulate LAPD activity. LAPD activity was determined as the maximal decrease compared to the maximal Ca 2+ signal amplitude after NKH477 addition. Data are shown as individual data points (each data point represents and independent experiment and corresponds to the average of a duplicate or triplicate measurement) and mean ± S.D., p-values calculated using unpaired, two-sided Student's t-test compared to Sstr3-eGFP are indicated. All HEK-293 cells were non-ciliated.

Techniques Used: Activity Assay, Binding Assay, Activation Assay, Laser Capture Microdissection, Expressing, Negative Control, Transfection, Incubation

Direct ciliary targeting of optogenetic tools impairs protein function. ( A ) Localization of mNphp3(201)-bPAC-mCherry to primary cilia. mIMCD-3 cells expressing mNphp3(201)-bPAC-mCherry were labeled with an anti-acetylated tubulin antibody (cyan, ciliary marker) and with DAPI (blue) to label the DNA. The box indicates the position of the magnified view shown at the bottom right. Red arrow indicates the direction and the length of the shift of the respective fluorescence channel. Scale bar: 10 μm. ( B ) Localization of mNphp3(201)-LAPD-mCherry to primary cilia. mIMCD-3 cells expressing mNphp3(201)- LAPD-mCherry were labeled with an anti-acetylated tubulin antibody (cyan, ciliary marker) and DAPI (blue) to label the DNA. The box indicates the position of the magnified view shown at the bottom right. Red arrow indicates the direction and the length of the shift of the respective red channel. Scale bar: 10 μm. ( C ) Assays to measure bPAC or LAPD activity using Ca 2+ imaging. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM) ( Wachten et al., 2006 ). Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylyl cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). ( D ) Quantification of bPAC activity. GFP-certified-FluoForte-loaded HEK-TM cells expressing mCherry only (grey), bPAC-mCherry (blue), or mNphp3(201)-bPAC-mCherry (cyan) were stimulated with 465 nm light pulses (1 mW/cm²) of different length and the increase in the intracellular Ca 2+ concentration was measured. To evoke a maximal Ca 2+ response, cells were stimulated with 2 μM ionomycin. Data are shown as mean ± SD (dotted lines) for the normalized fluorescence (F-F(baseline))/(F(ionomycin)-F(baseline))/fraction of mCherry-positive cells, n = 3 independent experiments (each data point represents the average of a duplicate or triplicate measurement). ( E ) Mean peak amplitudes of the Ca 2+ signal at 3–6 min after the first light pulse. Data are shown as individual data points and mean ± SD, n = 3. ( F ) Quantification of LAPD activity. Fluo4-AM-loaded HEK-TM cells expressing LAPD-mCherry (red) or mNphp3(201)-LAPD-mCherry (cyan) were incubated with 100 μM NKH477 during continuous 850 nm light stimulation (0.5 µW/cm²). At steady-state, light stimulation was switched to 690 nm (0.5 µW/cm²). NT: non-transfected cells (grey). Data are shown as mean ± SD (dotted lines) for the normalized fluorescence (F-F(baseline))/(F(ionomycin)-F(baseline)). ( G ) Mean decrease of the Ca 2+ signal after 690 nm light stimulation (fraction of maximum value after NKH477 increase), determined over 45 s at 3 min after switching to 690 nm. Data are shown as individual data points and mean ± SD, n = 4 independent experiments (each data point represents the average of a duplicate or triplicate measurement); p-values calculated using a paired, two-tailed t-test are indicated. NT: non-transfected cells.
Figure Legend Snippet: Direct ciliary targeting of optogenetic tools impairs protein function. ( A ) Localization of mNphp3(201)-bPAC-mCherry to primary cilia. mIMCD-3 cells expressing mNphp3(201)-bPAC-mCherry were labeled with an anti-acetylated tubulin antibody (cyan, ciliary marker) and with DAPI (blue) to label the DNA. The box indicates the position of the magnified view shown at the bottom right. Red arrow indicates the direction and the length of the shift of the respective fluorescence channel. Scale bar: 10 μm. ( B ) Localization of mNphp3(201)-LAPD-mCherry to primary cilia. mIMCD-3 cells expressing mNphp3(201)- LAPD-mCherry were labeled with an anti-acetylated tubulin antibody (cyan, ciliary marker) and DAPI (blue) to label the DNA. The box indicates the position of the magnified view shown at the bottom right. Red arrow indicates the direction and the length of the shift of the respective red channel. Scale bar: 10 μm. ( C ) Assays to measure bPAC or LAPD activity using Ca 2+ imaging. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca 2+ (HEK-TM) ( Wachten et al., 2006 ). Light-dependent activation of bPAC increases intracellular cAMP levels, leading to a Ca 2+ influx, which was quantified using a fluorescent Ca 2+ dye (GFP-certified FluoForte). To measure LAPD activity, HEK-TM cells were pre-stimulated with 100 μM NKH477 to activate transmembrane adenylyl cyclases (AC), thus increasing cAMP levels. Ca 2+ influx was detected by a Ca 2+ dye (Fluo4-AM). ( D ) Quantification of bPAC activity. GFP-certified-FluoForte-loaded HEK-TM cells expressing mCherry only (grey), bPAC-mCherry (blue), or mNphp3(201)-bPAC-mCherry (cyan) were stimulated with 465 nm light pulses (1 mW/cm²) of different length and the increase in the intracellular Ca 2+ concentration was measured. To evoke a maximal Ca 2+ response, cells were stimulated with 2 μM ionomycin. Data are shown as mean ± SD (dotted lines) for the normalized fluorescence (F-F(baseline))/(F(ionomycin)-F(baseline))/fraction of mCherry-positive cells, n = 3 independent experiments (each data point represents the average of a duplicate or triplicate measurement). ( E ) Mean peak amplitudes of the Ca 2+ signal at 3–6 min after the first light pulse. Data are shown as individual data points and mean ± SD, n = 3. ( F ) Quantification of LAPD activity. Fluo4-AM-loaded HEK-TM cells expressing LAPD-mCherry (red) or mNphp3(201)-LAPD-mCherry (cyan) were incubated with 100 μM NKH477 during continuous 850 nm light stimulation (0.5 µW/cm²). At steady-state, light stimulation was switched to 690 nm (0.5 µW/cm²). NT: non-transfected cells (grey). Data are shown as mean ± SD (dotted lines) for the normalized fluorescence (F-F(baseline))/(F(ionomycin)-F(baseline)). ( G ) Mean decrease of the Ca 2+ signal after 690 nm light stimulation (fraction of maximum value after NKH477 increase), determined over 45 s at 3 min after switching to 690 nm. Data are shown as individual data points and mean ± SD, n = 4 independent experiments (each data point represents the average of a duplicate or triplicate measurement); p-values calculated using a paired, two-tailed t-test are indicated. NT: non-transfected cells.

Techniques Used: Expressing, Labeling, Marker, Fluorescence, Activity Assay, Imaging, Binding Assay, Activation Assay, Concentration Assay, Incubation, Transfection, Two Tailed Test

18) Product Images from "Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6) *"

Article Title: Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.534156

Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p
Figure Legend Snippet: Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p

Techniques Used: Transfection, Construct, Confocal Microscopy

19) Product Images from "Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium"

Article Title: Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium

Journal: PLoS ONE

doi: 10.1371/journal.pone.0206276

TRPV4-mediated increase in cytosolic Ca 2+ ([Ca 2+ ] i ) in mouse primary colonic epithelial cells and CCD 841 cells. (A) [Ca 2+ ] i changes in response to the TRPV4 specific agonist, GSK101 (GSK, 30 nM), in WT or TRPV4-KO (V4KO) primary colonic epithelial cells (mean ± SEM). Ionomycin (iono) was used as a positive control. Bars indicate the period of chemical application. Significant increases in [Ca 2+ ] i at 90 seconds were observed in WT than V4KO (p
Figure Legend Snippet: TRPV4-mediated increase in cytosolic Ca 2+ ([Ca 2+ ] i ) in mouse primary colonic epithelial cells and CCD 841 cells. (A) [Ca 2+ ] i changes in response to the TRPV4 specific agonist, GSK101 (GSK, 30 nM), in WT or TRPV4-KO (V4KO) primary colonic epithelial cells (mean ± SEM). Ionomycin (iono) was used as a positive control. Bars indicate the period of chemical application. Significant increases in [Ca 2+ ] i at 90 seconds were observed in WT than V4KO (p

Techniques Used: Positive Control

20) Product Images from "TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis"

Article Title: TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0127480

Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].
Figure Legend Snippet: Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells. (A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) n = 54 (TMEM203 siRNA)].

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Mouse Assay, Microscopy, Transfection

Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .
Figure Legend Snippet: Intracellular store calcium flux and store operated calcium entry kinetics in testicular cells from WT and Tmem203 null mice. Flou3 and Fura red loaded testicular cells prepared from WT and Tmem203 null mice were analyzed by flow cytometry to follow cytosolic calcium kinetics in the gated predominately round spermatids population. Intracellular store calcium flux was measured by recording Flou3 calcium bound to Fura red calcium free ratio in the presence of 1mM EGTA in response to SERCA inhibitor- Thapsigargin (A); Calcium ionophore—Ionomycin (B); Similarly the store operated calcium entry kinetics was followed in WT and Tmem203 null testicular round spermatids gated population by depleting the stores by Thapsigargin (C) or Ionomycin (D) followed with addition of 2mM CaCl 2 .

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.
Figure Legend Snippet: TMEM203 interacts with regulators of ER calcium stores and overexpression depletes ER calcium stores. (A) Confocal analysis of HeLa cells transiently expressing TMEM203-GFP with organelle specific markers for ER (top:Calreticulin-RFP), Mitochondria (middle:BDHA-RFP) or plasma membrane (bottom:LCK-RFP). Separation or colocalization of TMEM203-GFP and organelle marker(s) were visualized by the linescan function of MetaMorph: the fluorescence intensity of each pixel of the line of interest (white lines ~ 75 μm) is shown as a xy-graph for the corresponding green and red channels. The line scan shows that TMEM203-GFP predominately overlapped with the ER marker. (Representative of ~ 50 cells from 2 independent experiments). Note, we cannot rule out that TMEM203 is completely absent from the the mitochondria. (B) Western analysis of complexes immune-precipitated TMEM203-Flag from HEK293 cells with indicated antibodies shows specific interaction with endogenous STIM1, IP3R and SERCA2. (Representative of atleast 2 independent experiments). (C) pTUNE-TMEM203-293cells were treated with the indicated dose of IPTG for 48 hrs to induce TMEM203 expression. Levels of TMEM203-Flag protein were detected by western blot. (D) These IPTG induced cells were subjected to Indo-1 based calcium flux measurements by flow cytometry by first treating with thapsigargin (TG) and EGTA. (E) As in (D) but the cells were treated with Ionomycin. (F) As in (D) but following TG treatment CaCl 2 was added to record SOCE.

Techniques Used: Over Expression, Expressing, Marker, Fluorescence, Western Blot, Flow Cytometry, Cytometry

21) Product Images from "Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata"

Article Title: Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata

Journal: PLoS ONE

doi: 10.1371/journal.pone.0139107

Frequency dependence of electrically-evoked calcium responses in neurites. (A) Overlay of Ca 2+ responses in a single neurite in response to sequential stimulation with 1, 5, 10, 20, 50 and 100 pulses over 1 second (grey bar). (B) Pooled data for average peak calcium response (ΔF/F 0 , black markers) as a function of stimulus frequency with an exponential regression fit (red line). Averaged data derives from recordings from 30 neurites excepting data for 0 Pulses (n = 8), 1 Pulse (n = 29) and 50 Pulses (n = 19). Spatiotemporal profile of fluorescent calcium signals along the terminal 200μm of a single neurite (rightmost inset) in response to stimulation with 1 current pulse (left panels), 5 pulses/s (centre panels) and 10 pulses/s (right panels). Fluorescent images were acquired at 25 Hz. The upper (blue) panels show fluorescence intensity coded as color along the length of the neurite (ordinate) and as a function of time (abscissa). Lower panels show the average fluorescence signal (ΔF/F 0 , open markers) determined from all pixels along the neurite ROI as a function of time. Individual electrical stimuli (1ms, 40mA) are shown as vertical grey lines. (C) (D) Fluorescence images of a neurite before (top), during electrical stimulation (centre) and during application of ionomycin (10μm) (bottom). (E) Calcium responses for the neurite in panel D as a function of time (upper) and spatiotemporally (lower) in response to electrical stimulation (20Hz, left) and ionomycin (10μM, right).
Figure Legend Snippet: Frequency dependence of electrically-evoked calcium responses in neurites. (A) Overlay of Ca 2+ responses in a single neurite in response to sequential stimulation with 1, 5, 10, 20, 50 and 100 pulses over 1 second (grey bar). (B) Pooled data for average peak calcium response (ΔF/F 0 , black markers) as a function of stimulus frequency with an exponential regression fit (red line). Averaged data derives from recordings from 30 neurites excepting data for 0 Pulses (n = 8), 1 Pulse (n = 29) and 50 Pulses (n = 19). Spatiotemporal profile of fluorescent calcium signals along the terminal 200μm of a single neurite (rightmost inset) in response to stimulation with 1 current pulse (left panels), 5 pulses/s (centre panels) and 10 pulses/s (right panels). Fluorescent images were acquired at 25 Hz. The upper (blue) panels show fluorescence intensity coded as color along the length of the neurite (ordinate) and as a function of time (abscissa). Lower panels show the average fluorescence signal (ΔF/F 0 , open markers) determined from all pixels along the neurite ROI as a function of time. Individual electrical stimuli (1ms, 40mA) are shown as vertical grey lines. (C) (D) Fluorescence images of a neurite before (top), during electrical stimulation (centre) and during application of ionomycin (10μm) (bottom). (E) Calcium responses for the neurite in panel D as a function of time (upper) and spatiotemporally (lower) in response to electrical stimulation (20Hz, left) and ionomycin (10μM, right).

Techniques Used: Fluorescence

22) Product Images from "Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury"

Article Title: Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.6008-11.2012

Strong VGCC activation induces lower Ca 2+ elevations and less death than NMDAR activation A. Time course of 90 mM K + -induced free Ca 2+ elevations (fura-2FF) in individual 14 DIV hippocampal neurons (gray traces) and their average (red; n =46). Incubation medium was 90 mM KCl, 1μM Bay K 8644, 20 μM MK-801, 10 μM CNQX in 10 mM Ca 2+ -containing HBSS. Blue traces – Average Ca 2+ elevations induced by 100 μM (dark blue; n =49) and 10 μM (light blue; n =47) NMDA; Orange trace – Average Ca 2+ response induced by high K + in the presence of 10 μM nimodipine ( n =35). B. Representative Ca 2+ traces from experiments in which Ca 2+ entry was induced by 100 μM NMDA (as in panel A), 10 μM ionomycin (Iono, n =30), or reverse mode NCX ( n =40; see Methods for protocol). C. Left panel – Maximal sustained concentration of free Ca 2+ in individual neurons 30 min after the activation of Ca 2+ uptake as in panels A and B); right panel – Neuronal death rate 24 h after the same stimuli. No significant cell death was observed in controls without stimulation. D. Concentrations of total calcium in the cytoplasm and mitochondria of neurons immediately after 30 min of stimulation as described in panels A and B. Data are mean ± SEM, as measured by EPMA. E. Time courses of Rh-123 relative fluorescence (F/F 0 ) show no significant changes of MMP in 14 DIV hippocampal neurons induced by 90 K + depolarization ( n =23) and typical strong MMP depolarization induced by NMDA (100 μM; n =26). F. Left panel – Representative traces of NO production (DAF-FM fluorescence; F/F 0 ) induced by 100 μM NMDA ( n =19) alone or in the presence 100 μM L-NAME ( n =14), or by 90 mM K + depolarization ( n =16). Right panel – Neuronal death rate 24 h after NMDA stimulation is reduced by pharmacological NOS inhibition (L-NAME) or by disrupting the NMDAR/nNOS linkage using a chemical LTD protocol (see Methods).
Figure Legend Snippet: Strong VGCC activation induces lower Ca 2+ elevations and less death than NMDAR activation A. Time course of 90 mM K + -induced free Ca 2+ elevations (fura-2FF) in individual 14 DIV hippocampal neurons (gray traces) and their average (red; n =46). Incubation medium was 90 mM KCl, 1μM Bay K 8644, 20 μM MK-801, 10 μM CNQX in 10 mM Ca 2+ -containing HBSS. Blue traces – Average Ca 2+ elevations induced by 100 μM (dark blue; n =49) and 10 μM (light blue; n =47) NMDA; Orange trace – Average Ca 2+ response induced by high K + in the presence of 10 μM nimodipine ( n =35). B. Representative Ca 2+ traces from experiments in which Ca 2+ entry was induced by 100 μM NMDA (as in panel A), 10 μM ionomycin (Iono, n =30), or reverse mode NCX ( n =40; see Methods for protocol). C. Left panel – Maximal sustained concentration of free Ca 2+ in individual neurons 30 min after the activation of Ca 2+ uptake as in panels A and B); right panel – Neuronal death rate 24 h after the same stimuli. No significant cell death was observed in controls without stimulation. D. Concentrations of total calcium in the cytoplasm and mitochondria of neurons immediately after 30 min of stimulation as described in panels A and B. Data are mean ± SEM, as measured by EPMA. E. Time courses of Rh-123 relative fluorescence (F/F 0 ) show no significant changes of MMP in 14 DIV hippocampal neurons induced by 90 K + depolarization ( n =23) and typical strong MMP depolarization induced by NMDA (100 μM; n =26). F. Left panel – Representative traces of NO production (DAF-FM fluorescence; F/F 0 ) induced by 100 μM NMDA ( n =19) alone or in the presence 100 μM L-NAME ( n =14), or by 90 mM K + depolarization ( n =16). Right panel – Neuronal death rate 24 h after NMDA stimulation is reduced by pharmacological NOS inhibition (L-NAME) or by disrupting the NMDAR/nNOS linkage using a chemical LTD protocol (see Methods).

Techniques Used: Activation Assay, Incubation, Concentration Assay, Fluorescence, Inhibition

Related Articles

Staining:

Article Title: Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations
Article Snippet: .. Cytokine expression staining was performed after culture with Phorbol-12,13-dibutyrate (1 μg/mL, Tocris Bioscience), Ionomycin calcium salt (1.5 μg/mL Tocris Bioscience) and Brefeldin A (4 μg/mL, Tocris Bioscience), and fixation with Foxp3/Transcription Factor Staining Buffer (Thermofisher). .. Cells were stained with anti-IL-4 (eBioscience, AB_469494) and anti-IFNγ (eBioscience, AB_469680).

other:

Article Title: The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs
Article Snippet: Chemical and drugs DAMGO, U50488H, SNC80, CGS21680, AVP (arginine vasopressin), IBMX, Forskolin, stromal cell-derived factor 1 (SDF-1 or CXCL12), carbamoylcholine chloride (carbachol), quinpirole hydrochloride, SKF 81297, ionomycin calcium salt, were purchased from Tocris Bioscience (Bristol, UK) and diluted in DMSO (diméthylsulfoxyde) at 10−2 M (except SDF1 at 125 µM and IBMX at 200 mM) for frozen stock aliquots and coelenterazine H substrate from Interchim (Montluçon, France) was diluted in 100% ethanol and kept at −20°C.

Positive Control:

Article Title: GDE2-Dependent Activation of Canonical Wnt Signaling in Neurons Regulates Oligodendrocyte Maturation
Article Snippet: .. At the end of image acquisition, 2 μM ionomycin (Tocris 1704) was added into each well as a positive control. .. Live recorded images of spontaneous calcium activity were acquired with epifluorescence microscope (Keyence BZ-X710) under 10X objective.

Article Title: Probing Internalization Effects and Biocompatibility of Ultrasmall Zirconium Metal-Organic Frameworks UiO-66 NP in U251 Glioblastoma Cancer Cells
Article Snippet: .. A positive control pressure perfused a solution of ionomycin calcium salt (Tocris Bioscience, Bristol, UK) for about 7 min. .. The estimation of intracellular free Ca2+ concentration was reported as change of the ratio between fluorescence emission at 510 nm, obtained with 340 and 380-nm excitation wavelengths (optical filters and dichroic beam splitter were from Lambda DG4, Shutter Instruments, Novato, CA, USA).

Expressing:

Article Title: Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations
Article Snippet: .. Cytokine expression staining was performed after culture with Phorbol-12,13-dibutyrate (1 μg/mL, Tocris Bioscience), Ionomycin calcium salt (1.5 μg/mL Tocris Bioscience) and Brefeldin A (4 μg/mL, Tocris Bioscience), and fixation with Foxp3/Transcription Factor Staining Buffer (Thermofisher). .. Cells were stained with anti-IL-4 (eBioscience, AB_469494) and anti-IFNγ (eBioscience, AB_469680).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Tocris m ionomycin
    H7 and Sta permit translocation of GFP-CaMKII in heterologous expression systems. (A) Representative time-course of stimulation-induced GFP-CaMKII translocation to GluN2B in HEK293 cells. <t>Ionomycin-stimulation</t> induces distinctive puncta and increased
    M Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m ionomycin/product/Tocris
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    m ionomycin - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    94
    Tocris ionomycin
    High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with <t>ionomycin</t> (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P
    Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Tocris
    Average 94 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    H7 and Sta permit translocation of GFP-CaMKII in heterologous expression systems. (A) Representative time-course of stimulation-induced GFP-CaMKII translocation to GluN2B in HEK293 cells. Ionomycin-stimulation induces distinctive puncta and increased

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.113.089045

    Figure Lengend Snippet: H7 and Sta permit translocation of GFP-CaMKII in heterologous expression systems. (A) Representative time-course of stimulation-induced GFP-CaMKII translocation to GluN2B in HEK293 cells. Ionomycin-stimulation induces distinctive puncta and increased

    Article Snippet: Following this treatment, the cells were either stimulated with 10 μ M ionomycin (Tocris) or mock and then incubated at room temperature for 5 minutes.

    Techniques: Translocation Assay, Expressing

    H7 and Sta inhibit CaMKII enzymatic activity within cells. To assess inhibition of CaMKII enzymatic activity within cells, HEK293 cells were cotransfected with CaMKII and GluA1. These cells were incubated with inhibitor and then stimulated with ionomycin

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.113.089045

    Figure Lengend Snippet: H7 and Sta inhibit CaMKII enzymatic activity within cells. To assess inhibition of CaMKII enzymatic activity within cells, HEK293 cells were cotransfected with CaMKII and GluA1. These cells were incubated with inhibitor and then stimulated with ionomycin

    Article Snippet: Following this treatment, the cells were either stimulated with 10 μ M ionomycin (Tocris) or mock and then incubated at room temperature for 5 minutes.

    Techniques: Activity Assay, Inhibition, Incubation

    AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Journal: PLoS ONE

    Article Title: mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

    doi: 10.1371/journal.pone.0154564

    Figure Lengend Snippet: AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Article Snippet: To determine the percentage of TH1 and TH17 cells, mononuclear cells were stimulated with PMA (50ng/mL, sigma) and Ionomycin (1μg/mL, Tocris) and BFA (1:1000, eBioscience) for 6 hours.

    Techniques: In Vitro, Isolation, Mouse Assay, Labeling, Staining, Expressing

    Time course of the effects of [Ca 2+ ] i -elevating stimuli on ERK1/2 phosphorylation in salivary gland cell lines. HSY cells (A) and Par-C10 cells (B) were exposed to TG (1 µM) and ionomycin (1 µM) for various times, as indicated. Values shown are for ERK1/2 phosphorylations (normalized to total ERK2) relative to basal conditions.

    Journal: PLoS ONE

    Article Title: Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells

    doi: 10.1371/journal.pone.0072881

    Figure Lengend Snippet: Time course of the effects of [Ca 2+ ] i -elevating stimuli on ERK1/2 phosphorylation in salivary gland cell lines. HSY cells (A) and Par-C10 cells (B) were exposed to TG (1 µM) and ionomycin (1 µM) for various times, as indicated. Values shown are for ERK1/2 phosphorylations (normalized to total ERK2) relative to basal conditions.

    Article Snippet: Thapsigargin (1138), ionomycin (1704), PP2 (1407), GF109203X (0741), Go6976 (2293), and SKF96363 (1147) were purchased from Tocris Bioscience.

    Techniques:

    Time course of the effects of [Ca 2+ ] i -elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells. A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE 2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p

    Journal: PLoS ONE

    Article Title: Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells

    doi: 10.1371/journal.pone.0072881

    Figure Lengend Snippet: Time course of the effects of [Ca 2+ ] i -elevating and other agents on ERK1/2 phosphorylation in rat parotid acinar cells. A. Comparison of effects of carbachol (10 µM), arachidonic acid (8 µM), TG (1 µM), and ionomycin (1 µM). B. Effect of PGE 2 (10 µM). C. Quantitative comparison of multiple agents on ERK1/2 phosphorylation relative to basal. Number of individual experiments is indicated at bottom of the bars. ***p

    Article Snippet: Thapsigargin (1138), ionomycin (1704), PP2 (1407), GF109203X (0741), Go6976 (2293), and SKF96363 (1147) were purchased from Tocris Bioscience.

    Techniques:

    High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with ionomycin (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P

    Journal: Frontiers in Immunology

    Article Title: Lysosomal Ca2+ Signaling Regulates High Glucose-Mediated Interleukin-1β Secretion via Transcription Factor EB in Human Monocytic Cells

    doi: 10.3389/fimmu.2017.01161

    Figure Lengend Snippet: High glucose (HG) upregulated transcription factor EB (TFEB) expression and lysosomal Ca 2+ -dependent TFEB nuclear translocation in U937 cells. (A) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm under low glucose (LG; 5.5 mM glucose), mannitol (Ma; 30 mM), or HG (10, 20, 30 mM glucose for 48 h) in U937 cells. The relative expression of TFEB was normalized to representative controls (Histone H3/GAPDH) ( n = 4). (B) Relative gene expression of TFEB under LG, Ma, or HG (10, 20, and 30 mM glucose for 48 h) in U937 cells ( n = 5). (C) Immunofluorescence images and representative graph showing the nuclear translocation of TFEB in U937 cells that were pre-treated with ionomycin (10 µM), U18666A (2 µg/ml), or thapsigargin (TG; 1 µM) under HG (30 mM glucose for 48 h). The graph represented the percentage of the cells with nuclear translocation of TFEB ( n = 4). (D) Representative immunoblots and graphs for TFEB and Histone H3 expressions in the nucleus, and TFEB and GAPDH expressions in the cytoplasm after stimulation with H 2 O 2 (400 µM), ionomycin (1 µM), glycyl- l -phenylalanine-beta-naphthylamide (GPN; 400 µM), nicotinic acid adenine dinucleotide phosphate (NAADP; 1 mM), or TG (400 nM) in U937 cells. The relative protein expression of TFEB was normalized to representative controls (histone H3/GAPDH) ( n = 4). Data were shown as mean ± SEM. (A–C) * P

    Article Snippet: Bafilomycin A1 and GPN were from Santa Cruz Biotechnology, while BAPTA-AM, cyclosporin A, FK506, ionomycin, nicotinic acid adenine dinucleotide phosphate (NAADP), trans -Ned-19 (Ned-19), and U18666A were from Tocris Biosciences, USA.

    Techniques: Expressing, Translocation Assay, Western Blot, Immunofluorescence