Structured Review

Merck KGaA ionomycin
Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore <t>ionomycin</t> was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.
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1) Product Images from "Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion"

Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063521

Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.
Figure Legend Snippet: Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

Techniques Used: Chemotaxis Assay, Purification, Migration, Flow Cytometry, Cytometry, Negative Control, Positive Control

2) Product Images from "Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation"

Article Title: Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation

Journal: Scientific Reports

doi: 10.1038/s41598-017-15668-z

Accelerated lipid oxidation is essential for SSZ-induced NETosis. ( a – d ) Mouse neutrophils were stimulated with various concentrations of PMA ( a , b ) or ionomycin ( c , d ) in the presence or absence of 1 mM SSZ for 1 h. C11-Bodipy 581/591 was then added. ( a , c ) The accumulation of lipid oxidation was analyzed using flow cytometry. ( b , d ) Average mean fluorescent intensity (MFI) of C11-Bodipy analysis with s.d. of triplicated samples are shown. * P
Figure Legend Snippet: Accelerated lipid oxidation is essential for SSZ-induced NETosis. ( a – d ) Mouse neutrophils were stimulated with various concentrations of PMA ( a , b ) or ionomycin ( c , d ) in the presence or absence of 1 mM SSZ for 1 h. C11-Bodipy 581/591 was then added. ( a , c ) The accumulation of lipid oxidation was analyzed using flow cytometry. ( b , d ) Average mean fluorescent intensity (MFI) of C11-Bodipy analysis with s.d. of triplicated samples are shown. * P

Techniques Used: Flow Cytometry, Cytometry

3) Product Images from "IKK and NF-?B-mediated regulation of Claspin impacts on ATR checkpoint function"

Article Title: IKK and NF-?B-mediated regulation of Claspin impacts on ATR checkpoint function

Journal: The EMBO Journal

doi: 10.1038/emboj.2010.171

Claspin mRNA is regulated independently of the cell cycle but Claspin is responsive to NF-κB activation. ( A ) U2OS cells were depleted of IKKα, IKKβ, Claspin or Cyclin D1 using siRNA. WCLs were subjected to western blot analysis for the levels of the indicated proteins. ( B ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of IKKα, IKKβ, Claspin or Cyclin D1 by siRNA. ANOVA t -tests were performed on the means, and the P -values were calculated. ** P ⩽0.01 and *** P ⩽0.001. ( C ) U2OS cells were synchronized at G1/S using a double-thymidine block. Thymidine was subsequently washed and cells were released for the indicated times. Cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared from these cells and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( D ) U2OS cells were synchronized at prometaphase using nocodazole. Nocodazole was subsequently washed and cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( E ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of β-TrCP by siRNA. ( F ) U2OS cells were treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h. One hour prior to harvesting, cells were treated with UV (40 J/m 2 ) as indicated. WCLs were prepared and Claspin levels were analysed by western blot.
Figure Legend Snippet: Claspin mRNA is regulated independently of the cell cycle but Claspin is responsive to NF-κB activation. ( A ) U2OS cells were depleted of IKKα, IKKβ, Claspin or Cyclin D1 using siRNA. WCLs were subjected to western blot analysis for the levels of the indicated proteins. ( B ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of IKKα, IKKβ, Claspin or Cyclin D1 by siRNA. ANOVA t -tests were performed on the means, and the P -values were calculated. ** P ⩽0.01 and *** P ⩽0.001. ( C ) U2OS cells were synchronized at G1/S using a double-thymidine block. Thymidine was subsequently washed and cells were released for the indicated times. Cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared from these cells and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( D ) U2OS cells were synchronized at prometaphase using nocodazole. Nocodazole was subsequently washed and cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( E ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of β-TrCP by siRNA. ( F ) U2OS cells were treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h. One hour prior to harvesting, cells were treated with UV (40 J/m 2 ) as indicated. WCLs were prepared and Claspin levels were analysed by western blot.

Techniques Used: Activation Assay, Western Blot, Quantitative RT-PCR, Blocking Assay, Flow Cytometry, Cytometry

Loss or inhibition of IKKβ disrupts Chk1 phosphorylation in response to UV. ( A ) U2OS cells were depleted of IKKα, IKKβ, IKKγ or Claspin with siRNA. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( B ) U2OS cell were pretreated with the IKK inhibitor, Bay 11-7082, for 24 h, as indicated. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( C ) U2OS cells were depleted of the NF-κB transcription factors RelA, RelB, c-Rel, p50 or p52 with siRNA. Cells were treated with UV as in ( A ) and WCLs prepared analysed by western blot using the indicated antibodies. ( D ) U2OS cells were transfected with control or constitutive active IKKβ (left panel) or treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h (right panel) prior to treatment with UV (40 J/m 2 ) as indicated. WCLs were prepared and analysed by western blot using the indicated antibodies. ( E ) U2OS cells were transfected with a non-targeting siRNA or a siRNA to deplete IKKβ. c-Rel was then re-introduced into these cells by transient transfection, and cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies. ( F ) U2OS cells were co-transfected with a non-targeting siRNA or a siRNA to deplete IKKβ, along with a DNA construct to re-introduce Claspin. Cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies.
Figure Legend Snippet: Loss or inhibition of IKKβ disrupts Chk1 phosphorylation in response to UV. ( A ) U2OS cells were depleted of IKKα, IKKβ, IKKγ or Claspin with siRNA. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( B ) U2OS cell were pretreated with the IKK inhibitor, Bay 11-7082, for 24 h, as indicated. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( C ) U2OS cells were depleted of the NF-κB transcription factors RelA, RelB, c-Rel, p50 or p52 with siRNA. Cells were treated with UV as in ( A ) and WCLs prepared analysed by western blot using the indicated antibodies. ( D ) U2OS cells were transfected with control or constitutive active IKKβ (left panel) or treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h (right panel) prior to treatment with UV (40 J/m 2 ) as indicated. WCLs were prepared and analysed by western blot using the indicated antibodies. ( E ) U2OS cells were transfected with a non-targeting siRNA or a siRNA to deplete IKKβ. c-Rel was then re-introduced into these cells by transient transfection, and cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies. ( F ) U2OS cells were co-transfected with a non-targeting siRNA or a siRNA to deplete IKKβ, along with a DNA construct to re-introduce Claspin. Cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies.

Techniques Used: Inhibition, Western Blot, Transfection, Construct, Introduce

4) Product Images from "Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection"

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection

Journal: Nature Communications

doi: 10.1038/s41467-018-05519-4

A129 mice 4-week-old survive to ZIKV PE243 and evoke a robust immune response. Three- and 4-week-old A129 mice were inoculated intravenously with 2 × 10 5 PFU of ZIKV strain PE243. Uninfected mice were used as control. a , b Body weight gain ( a ) and lethality ( b ) of 3- ( n = 9) or 4-week-old A129 mice ( n = 15) infected with ZIKV PE243 were monitored for up to 15 days postinfection. c ZIKV RNA copies in the spleen, kidney, liver, and brain determined by qRT-PCR at days 3, 5, and 7 postinfection of 3- ( n = 3–8) or 4-week-old mice ( n = 4–6). Results are expressed as RNA equivalent/copies and normalized by GAPDH. Flow cytometry were performed on splenocytes from uninfected ( n = 4–8) or 4-week-old mice at day 7 postinfection ( n = 4–15). For cytokines and cytotoxic factors detection, splenocytes were restimulated in vitro with PMA and ionomycin in the presence of brefeldin A during 4 h. d Representative dot plot and frequency of CD62L − CD44 + (effector) among CD8 + . e Representative counter plot and frequency of IFNγ, IL-2, CD107a, granzyme B (GrzmB), and perforin (Prfn) among CD8 + T cells. f Representative dot plot and frequency of CD62L - CD44 + (effector) among CD4 + . g Representative counter plot and frequency of I IFNγ, IL-4, and IL-17 among CD4 + T cells. h Representative counter plot and frequency of CD25 + Foxp3 + or CXCR5 + PD-1 + and CXCR5 + PD-1 + IFNγ + among CD4 + T cells. i Representative counter plot and frequency of germinal center B cells among B220 + CD138 − and plasma cells. Results are shown as mean in d – i or as mean ± standard deviation in a and c . Data are representative of two independent experiments in a , b , and c , data are presented as a pool of two or three independent experiments in d – i . Survival data were analyzed by log rank test. Data were analyzed by Student’s t test in c–i. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001
Figure Legend Snippet: A129 mice 4-week-old survive to ZIKV PE243 and evoke a robust immune response. Three- and 4-week-old A129 mice were inoculated intravenously with 2 × 10 5 PFU of ZIKV strain PE243. Uninfected mice were used as control. a , b Body weight gain ( a ) and lethality ( b ) of 3- ( n = 9) or 4-week-old A129 mice ( n = 15) infected with ZIKV PE243 were monitored for up to 15 days postinfection. c ZIKV RNA copies in the spleen, kidney, liver, and brain determined by qRT-PCR at days 3, 5, and 7 postinfection of 3- ( n = 3–8) or 4-week-old mice ( n = 4–6). Results are expressed as RNA equivalent/copies and normalized by GAPDH. Flow cytometry were performed on splenocytes from uninfected ( n = 4–8) or 4-week-old mice at day 7 postinfection ( n = 4–15). For cytokines and cytotoxic factors detection, splenocytes were restimulated in vitro with PMA and ionomycin in the presence of brefeldin A during 4 h. d Representative dot plot and frequency of CD62L − CD44 + (effector) among CD8 + . e Representative counter plot and frequency of IFNγ, IL-2, CD107a, granzyme B (GrzmB), and perforin (Prfn) among CD8 + T cells. f Representative dot plot and frequency of CD62L - CD44 + (effector) among CD4 + . g Representative counter plot and frequency of I IFNγ, IL-4, and IL-17 among CD4 + T cells. h Representative counter plot and frequency of CD25 + Foxp3 + or CXCR5 + PD-1 + and CXCR5 + PD-1 + IFNγ + among CD4 + T cells. i Representative counter plot and frequency of germinal center B cells among B220 + CD138 − and plasma cells. Results are shown as mean in d – i or as mean ± standard deviation in a and c . Data are representative of two independent experiments in a , b , and c , data are presented as a pool of two or three independent experiments in d – i . Survival data were analyzed by log rank test. Data were analyzed by Student’s t test in c–i. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Flow Cytometry, Cytometry, In Vitro, Standard Deviation

5) Product Images from "Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis"

Article Title: Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

Journal: Scientific Reports

doi: 10.1038/s41598-017-07556-3

Identification of substrates specific for RHBDL2 ( A ). Strep tagged substrates were co-expressed with HA tagged forms of mouse RHBDL2, the four human rhomboids (R1/R2/R3/R4) and their corresponding inactivated forms where the catalytic serine residue was mutated to an alanine (SA). Twenty four hours after transfection, the medium was replaced by serum-free medium containing 10 µM broad spectrum matrix metalloprotease inhibitor BB94 to exclude shedding by these extracellular proteases. Forty eight hours after transfection, the media and cell lysates were harvested and analysed by immunoblotting. Cells were co-transfected with FLAG tagged prolactin as a secretion control and to confirm recovery of TCA precipitated proteins from the media. ( B ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 1 µM ionomycin, 1 µM ionomycin and 2 mM EGTA, or 1 µM ionomycin and 10 µM BB94. One hour after media replacement the media (upper panels) and cell lysate (lower panels) were harvested as previously described and analysed by immunoblotting. ( C ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 0.5 µM phorbol 12-myristate 13-acetate (PMA), the inactive analogue 4α-phorbol 12,13-didecanoate (4αPDD), or both 0.5 µM PMA and 10 µM BB94. One hour after media replacement the media and cell lysate were harvested as previously described and analysed by immunoblotting. Asterisks indicate samples which required treatment with PNGase F prior to SDS-PAGE to reduce smearing and improve resolution of bands on the gel.
Figure Legend Snippet: Identification of substrates specific for RHBDL2 ( A ). Strep tagged substrates were co-expressed with HA tagged forms of mouse RHBDL2, the four human rhomboids (R1/R2/R3/R4) and their corresponding inactivated forms where the catalytic serine residue was mutated to an alanine (SA). Twenty four hours after transfection, the medium was replaced by serum-free medium containing 10 µM broad spectrum matrix metalloprotease inhibitor BB94 to exclude shedding by these extracellular proteases. Forty eight hours after transfection, the media and cell lysates were harvested and analysed by immunoblotting. Cells were co-transfected with FLAG tagged prolactin as a secretion control and to confirm recovery of TCA precipitated proteins from the media. ( B ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 1 µM ionomycin, 1 µM ionomycin and 2 mM EGTA, or 1 µM ionomycin and 10 µM BB94. One hour after media replacement the media (upper panels) and cell lysate (lower panels) were harvested as previously described and analysed by immunoblotting. ( C ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 0.5 µM phorbol 12-myristate 13-acetate (PMA), the inactive analogue 4α-phorbol 12,13-didecanoate (4αPDD), or both 0.5 µM PMA and 10 µM BB94. One hour after media replacement the media and cell lysate were harvested as previously described and analysed by immunoblotting. Asterisks indicate samples which required treatment with PNGase F prior to SDS-PAGE to reduce smearing and improve resolution of bands on the gel.

Techniques Used: Transfection, SDS Page

6) Product Images from "ORP4L is essential for T-cell acute lymphoblastic leukemia cell survival"

Article Title: ORP4L is essential for T-cell acute lymphoblastic leukemia cell survival

Journal: Nature Communications

doi: 10.1038/ncomms12702

ORP4L sustains Ca 2+ -dependent bioenergetics in T-ALL cells. ( a , b ) Western blot analysis of PDH activation in Jurkat, Molt-4 and primary T-ALL cells with ORP4L knockdown ( a ) and overexpression ( b ). p-PDH/PDH expressed as fold change over control. ( c ) PDH activation (left), OCR (middle) and ATP levels (right) in Jurkat T-cells with ORP4L knockdown alone or in combination with shPLCβ3. ( d ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without U73122 (5 μM for 1 h) or XeC (2 μM for 1 h). ( e ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without ionomycin (2 mg l −1 for 1 h). ( f ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without BAPTA-AM (50 μM for 1 h). ( g ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without MCU agonist, kaempferol (2 μM, 30 min). ( h ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without MCU inhibitor, RU360 (5 μM, 30 min). The data represent mean±s.d. value from an experiment performed in triplicate. * P
Figure Legend Snippet: ORP4L sustains Ca 2+ -dependent bioenergetics in T-ALL cells. ( a , b ) Western blot analysis of PDH activation in Jurkat, Molt-4 and primary T-ALL cells with ORP4L knockdown ( a ) and overexpression ( b ). p-PDH/PDH expressed as fold change over control. ( c ) PDH activation (left), OCR (middle) and ATP levels (right) in Jurkat T-cells with ORP4L knockdown alone or in combination with shPLCβ3. ( d ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without U73122 (5 μM for 1 h) or XeC (2 μM for 1 h). ( e ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without ionomycin (2 mg l −1 for 1 h). ( f ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without BAPTA-AM (50 μM for 1 h). ( g ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without MCU agonist, kaempferol (2 μM, 30 min). ( h ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without MCU inhibitor, RU360 (5 μM, 30 min). The data represent mean±s.d. value from an experiment performed in triplicate. * P

Techniques Used: Western Blot, Activation Assay, Over Expression

7) Product Images from "Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion"

Article Title: Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-019-0463-y

NFATC1 is constitutively active in PC-3 cells. Flag-tagged NFATC1 or its mutated derivatives were transiently expressed in PC-3 prostate cancer cells. Untransfected (−) or mock-transfected cells were used as controls. a The endogenous or ectopic expression levels of NFATC1 were analysed by Western blotting with antibodies against NFATc1 or Flag, while ACTB staining was used as a loading control. b The endogenous NFAT activity of PC-3 cells was measured by luciferase assays, using transiently transfected reporters with wild-type (WT) or mutated (M) NFAT binding sites. Shown are mean luciferase activities from two independent experiments. c The effects of TPA and ionomycin on NFAT activity were measured by luciferase assays. Shown are luciferase activities of duplicate samples from one representative experiment. d Subcellular localizations of transiently expressed wild-type (WT) NFATC1, the constitutively active (mSRR) mutant and the dominant negative (DN) mutant were analysed by confocal microscopy after staining with anti-Flag antibody. Shown are average localization patterns from one experiment with three parallel samples. e The abilities of WT NFATC1 and the mSRR mutant to promote cell motility were analysed by wound healing assays from three parallel samples. Equivalent expression of these proteins was confirmed by Western blotting with anti-Flag antibody, while GAPDH staining was used as a loading control
Figure Legend Snippet: NFATC1 is constitutively active in PC-3 cells. Flag-tagged NFATC1 or its mutated derivatives were transiently expressed in PC-3 prostate cancer cells. Untransfected (−) or mock-transfected cells were used as controls. a The endogenous or ectopic expression levels of NFATC1 were analysed by Western blotting with antibodies against NFATc1 or Flag, while ACTB staining was used as a loading control. b The endogenous NFAT activity of PC-3 cells was measured by luciferase assays, using transiently transfected reporters with wild-type (WT) or mutated (M) NFAT binding sites. Shown are mean luciferase activities from two independent experiments. c The effects of TPA and ionomycin on NFAT activity were measured by luciferase assays. Shown are luciferase activities of duplicate samples from one representative experiment. d Subcellular localizations of transiently expressed wild-type (WT) NFATC1, the constitutively active (mSRR) mutant and the dominant negative (DN) mutant were analysed by confocal microscopy after staining with anti-Flag antibody. Shown are average localization patterns from one experiment with three parallel samples. e The abilities of WT NFATC1 and the mSRR mutant to promote cell motility were analysed by wound healing assays from three parallel samples. Equivalent expression of these proteins was confirmed by Western blotting with anti-Flag antibody, while GAPDH staining was used as a loading control

Techniques Used: Transfection, Expressing, Western Blot, Staining, Activity Assay, Luciferase, Binding Assay, Mutagenesis, Dominant Negative Mutation, Confocal Microscopy

8) Product Images from "Curcumin Suppresses T Cell Activation by Blocking Ca2+ Mobilization and Nuclear Factor of Activated T Cells (NFAT) Activation"

Article Title: Curcumin Suppresses T Cell Activation by Blocking Ca2+ Mobilization and Nuclear Factor of Activated T Cells (NFAT) Activation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.318733

Curcumin suppresses CD69 expression in activated T cells. A and B , curcumin suppresses CD69 expression in T cells. Jurkat ( A ) and freshly isolated human peripheral blood T cells ( B ) were stimulated with PMA (5 ng/ml) and ionomycin (1 μ m ) in the
Figure Legend Snippet: Curcumin suppresses CD69 expression in activated T cells. A and B , curcumin suppresses CD69 expression in T cells. Jurkat ( A ) and freshly isolated human peripheral blood T cells ( B ) were stimulated with PMA (5 ng/ml) and ionomycin (1 μ m ) in the

Techniques Used: Expressing, Isolation

Curcumin enhances the immunosuppressive activities of CsA. A , curcumin and CsA synergize to inhibit CD69 expression in activated T cells. Jurkat T cells were stimulated with PMA/ionomycin in the absence or the presence of either CsA alone or CsA plus
Figure Legend Snippet: Curcumin enhances the immunosuppressive activities of CsA. A , curcumin and CsA synergize to inhibit CD69 expression in activated T cells. Jurkat T cells were stimulated with PMA/ionomycin in the absence or the presence of either CsA alone or CsA plus

Techniques Used: Expressing

9) Product Images from "Revealing the Molecular Mechanism of Gastric Cancer Marker Annexin A4 in Cancer Cell Proliferation Using Exon Arrays"

Article Title: Revealing the Molecular Mechanism of Gastric Cancer Marker Annexin A4 in Cancer Cell Proliferation Using Exon Arrays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044615

Ca 2+ mediates the expression of RHAMM, phospho-AKT and p21. (A) Cells were treated with ionomycin to increase intracellular Ca 2+ levels, and the expression levels of ANXA4, LAMP2, RHAMM, phospho-AKT (Ser473), p21, and phospho-CDK1 (Thr161) were showed by immunoblotting. (B) The histogram shows the related levels of (A). The relative expressions of RHAMM ( P
Figure Legend Snippet: Ca 2+ mediates the expression of RHAMM, phospho-AKT and p21. (A) Cells were treated with ionomycin to increase intracellular Ca 2+ levels, and the expression levels of ANXA4, LAMP2, RHAMM, phospho-AKT (Ser473), p21, and phospho-CDK1 (Thr161) were showed by immunoblotting. (B) The histogram shows the related levels of (A). The relative expressions of RHAMM ( P

Techniques Used: Expressing

10) Product Images from "NFκB attenuates IL-5 production and upregulates T-box transcription factors in Th2-like T cells"

Article Title: NFκB attenuates IL-5 production and upregulates T-box transcription factors in Th2-like T cells

Journal: Cytotechnology

doi: 10.1007/s10616-013-9585-z

Rel transcriptionally suppresses IL-5 expression. Gata3-transduced DO11.10 T cell hybridoma was retrovirally transduced with pMXs-IG (control) or pMXs-RelA-IG ( Rela ), and IL-5 production in response to PMA + ionomycin + bt2cAMP
Figure Legend Snippet: Rel transcriptionally suppresses IL-5 expression. Gata3-transduced DO11.10 T cell hybridoma was retrovirally transduced with pMXs-IG (control) or pMXs-RelA-IG ( Rela ), and IL-5 production in response to PMA + ionomycin + bt2cAMP

Techniques Used: Expressing, Transduction

Gata3 transduction leads to IL-5 production in T cell hybridoma. a DO11.10 T cell hybridoma was retrovirally transduced with control pMXs-IG or pMXs-Gata3-IG ( Gata3 ), and IL-5 production in response to PMA/ionomycin/bt 2 cAMP was measured by intracellular
Figure Legend Snippet: Gata3 transduction leads to IL-5 production in T cell hybridoma. a DO11.10 T cell hybridoma was retrovirally transduced with control pMXs-IG or pMXs-Gata3-IG ( Gata3 ), and IL-5 production in response to PMA/ionomycin/bt 2 cAMP was measured by intracellular

Techniques Used: Transduction

11) Product Images from "The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells"

Article Title: The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192208

Interaction of CacyBP/SIP and S100A6 in a Ca2+-dependent manner in SW480 cells. CacyBP/SIP was precipitated by antibody against CacyBP/SIP, and the co-precipitation of S100A1 and S100A6 was analyzed by western blot analysis. The protein levels of CacyBP/SIP, S100A1, and S100A6 in cell lysates were analyzed by western blot analysis. Cells were treated with ionomycin (5 μmol/L) for 30 min. Data are representative immunoblots of three independent assays.
Figure Legend Snippet: Interaction of CacyBP/SIP and S100A6 in a Ca2+-dependent manner in SW480 cells. CacyBP/SIP was precipitated by antibody against CacyBP/SIP, and the co-precipitation of S100A1 and S100A6 was analyzed by western blot analysis. The protein levels of CacyBP/SIP, S100A1, and S100A6 in cell lysates were analyzed by western blot analysis. Cells were treated with ionomycin (5 μmol/L) for 30 min. Data are representative immunoblots of three independent assays.

Techniques Used: Western Blot

The effect BAPTA/AM on the nuclear localization of CacyBP/SIP induced by ionomycin in SW480 cells. (A) Effect of BAPTA/AM on ionomycin-stimulated nuclear translocation of endogenous CacyBP/SIP. Cells were treated with 5 μmol/L of ionomycin plus different concentrations of BAPTA/AM (0, 5, 10, and 25 μmol/L) for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m); nuclei were labeled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 μm. (B) The intensity of cytosolic free intracellular Ca 2+ fluorescence stimulated by 5 μmol/L of ionomycin plus different concentrations of BAPTA/AM (0, 5, 10, and 25 μmol/L) in SW480 cells. BAPTA/AM at concentrations of 10 μmol/L and 25 μmol/L totally abolished the nuclear translocation of CacyBP/SIP induced by 5 μmol/L of ionomycin. Cells were loaded with 20 μmol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. The Fluo-3 fluorescence intensity was reduced by 5 μmol/L of ionomycin plus 10 μmol/L and 25 μmol/L of BAPTA/AM. Error bars represent the mean±s.d.
Figure Legend Snippet: The effect BAPTA/AM on the nuclear localization of CacyBP/SIP induced by ionomycin in SW480 cells. (A) Effect of BAPTA/AM on ionomycin-stimulated nuclear translocation of endogenous CacyBP/SIP. Cells were treated with 5 μmol/L of ionomycin plus different concentrations of BAPTA/AM (0, 5, 10, and 25 μmol/L) for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m); nuclei were labeled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 μm. (B) The intensity of cytosolic free intracellular Ca 2+ fluorescence stimulated by 5 μmol/L of ionomycin plus different concentrations of BAPTA/AM (0, 5, 10, and 25 μmol/L) in SW480 cells. BAPTA/AM at concentrations of 10 μmol/L and 25 μmol/L totally abolished the nuclear translocation of CacyBP/SIP induced by 5 μmol/L of ionomycin. Cells were loaded with 20 μmol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. The Fluo-3 fluorescence intensity was reduced by 5 μmol/L of ionomycin plus 10 μmol/L and 25 μmol/L of BAPTA/AM. Error bars represent the mean±s.d.

Techniques Used: Translocation Assay, Immunostaining, Confocal Microscopy, Labeling, Fluorescence, Microscopy, Size-exclusion Chromatography

Effect of increased [Ca 2+ ]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells. (A) Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. Cells were treated with ionomycin for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. CacyBP/SIP was translocated to the perinuclear region in SW480 cells. After stimulation with an increasing amount of ionomycin (0, 1, 2, 5, 10 μmol/L) for 30 min at 37°C, SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m), and nuclei were labelled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 μm. (B) The intensity of cytosolic free intracellular Ca 2+ fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 μmol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 μmol/L and 10 μmol/L of ionomycin. SW480 cells were loaded with 20 μmol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. Ca 2+ concentration is increased by treatment with 2, 5, and 10 μmol/L of ionomycin ( ***P
Figure Legend Snippet: Effect of increased [Ca 2+ ]i on the subcellular localization of CacyBP/SIP in colon cancer SW480 cells. (A) Effect of different concentrations of ionomycin on the localization of endogenous CacyBP/SIP. Cells were treated with ionomycin for 30 min, followed by immunostaining using anti-CacyBP/SIP, and were imaged with confocal microscopy. CacyBP/SIP was translocated to the perinuclear region in SW480 cells. After stimulation with an increasing amount of ionomycin (0, 1, 2, 5, 10 μmol/L) for 30 min at 37°C, SW480 cells were fixed and immunostained using CacyBP/SIP MAb (panels a, d, g, j, and m), and nuclei were labelled with DAPI (panels b, e, h, k, and n). The merged images are shown in panels c, f, i, l, and o. The scale bar represents 50 μm. (B) The intensity of cytosolic free intracellular Ca 2+ fluorescence in SW480 cells treated with ionomycin (0, 1, 2, 5, 10 μmol/L). The Fluo-3 fluorescence intensity in SW480 cells reached a plateau at 5 μmol/L and 10 μmol/L of ionomycin. SW480 cells were loaded with 20 μmol/L of Fluo-3/AM for 45 min under a confocal microscope (495 nm). The fluorescence was captured every 2 sec and recorded for 3 min. (C) The bar chart shows the intracellular Fluo-3 intensity. Ca 2+ concentration is increased by treatment with 2, 5, and 10 μmol/L of ionomycin ( ***P

Techniques Used: Immunostaining, Confocal Microscopy, Fluorescence, Microscopy, Size-exclusion Chromatography, Concentration Assay

12) Product Images from "Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals"

Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

Journal: Neurochemical Research

doi: 10.1007/s11064-015-1663-5

Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p
Figure Legend Snippet: Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p

Techniques Used: Incubation

Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p
Figure Legend Snippet: Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p

Techniques Used: Incubation

[Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p
Figure Legend Snippet: [Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p

Techniques Used: Incubation

Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p
Figure Legend Snippet: Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p

Techniques Used: Activity Assay, Incubation

13) Product Images from "Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1"

Article Title: Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201506022

Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM thapsigargin in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P
Figure Legend Snippet: Stable and transient expression of σ1R inhibits SOCE. (A) Ca 2+ signals evoked by 1 µM thapsigargin in Ca 2+ -free HBS followed by restoration of 4 mM extracellular Ca 2+ in HEK wild-type cells treated with CPA (0.5 µM or 1 µM for 2.5 h) or HEK-σ1R cells. (B) Summary results show peak increases in [Ca 2+ ] c evoked by SOCE or by addition of ionomycin in Ca 2+ -free HBS ( n = 3). (C) Populations of fura 2–loaded cells were treated with thapsigargin (5 µM for 10 min) in nominally Ca 2+ -free HBS before addition of 5 mM MnCl 2 . Results show normalized fluorescence intensity (F/F 0 ) for six replicates. WT, wild type. (D) Summary results ( n = 3) show half-times (t 1/2 ) for fluorescence quenching from unstimulated cells (basal) and cells treated with thapsigargin (5 µM for 10 min) or ATP and carbachol (100 µM each for 3.5 min). (E) Typical images of HEK cells expressing NFAT-GFP before and 30 min after addition of 5 µM thapsigargin in normal HBS (top). Bar, 10 µm. Images of larger fields (bottom) show thapsigargin-treated HEK wild-type and HEK-σ1R cells. Asterisks indicate cells used for analysis. Bar, 20 µm. (F) Summary results show nuclear translocation of NFAT-GFP before and after treatment with thapsigargin (percentage of cells; six independent fields, with between 595 and 660 cells counted for each condition). *, P

Techniques Used: Expressing, Fluorescence, Translocation Assay

Ligands of σ1R modulate SOCE. (A–F) Populations of cells were treated with 25 µM (+)SKF10047 or 10 µM BD1047 before removal of extracellular Ca 2+ , addition of 5 µM thapsigargin, and then restoration of extracellular 4 mM Ca 2+ to CHO (A and B), HEK-σ1R (C and D), or wild-type HEK cells (E and F). Summary results (B, D, and F) show peak increases in [Ca 2+ ] c after restoration of extracellular Ca 2+ . The color codes in A apply to all panels (A–F). (G) Representative immunoblot from CHO cells transfected with control plasmid or plasmid encoding siRNA for σ1R (siσ1R). (H) Summary results show band intensities for the indicated proteins normalized to those from cells treated with control plasmid. (I) Ca 2+ signals evoked by addition of thapsigargin in Ca 2+ -free HBS and then restoration of extracellular Ca 2+ in CHO cells treated with siσ1R or control plasmid. (J) Summary shows peak [Ca 2+ ] c after restoration of extracellular Ca 2+ to thapsigargin-treated CHO cells treated with siσ1R or control plasmid. Cells were pretreated with 25 µM (+)SKF10047 or 10 µM BD1047, as indicated. (K and L) Effects of siσ1R or control plasmid and pretreatment with σ1R ligands on the Ca 2+ signals evoked by 5 µM ionomycin in Ca 2+ -free HBS. Typical traces (K) and summary results (L) are shown. Legends for L are the same as J. All summary results show mean ± SEM. n = 3. *, P
Figure Legend Snippet: Ligands of σ1R modulate SOCE. (A–F) Populations of cells were treated with 25 µM (+)SKF10047 or 10 µM BD1047 before removal of extracellular Ca 2+ , addition of 5 µM thapsigargin, and then restoration of extracellular 4 mM Ca 2+ to CHO (A and B), HEK-σ1R (C and D), or wild-type HEK cells (E and F). Summary results (B, D, and F) show peak increases in [Ca 2+ ] c after restoration of extracellular Ca 2+ . The color codes in A apply to all panels (A–F). (G) Representative immunoblot from CHO cells transfected with control plasmid or plasmid encoding siRNA for σ1R (siσ1R). (H) Summary results show band intensities for the indicated proteins normalized to those from cells treated with control plasmid. (I) Ca 2+ signals evoked by addition of thapsigargin in Ca 2+ -free HBS and then restoration of extracellular Ca 2+ in CHO cells treated with siσ1R or control plasmid. (J) Summary shows peak [Ca 2+ ] c after restoration of extracellular Ca 2+ to thapsigargin-treated CHO cells treated with siσ1R or control plasmid. Cells were pretreated with 25 µM (+)SKF10047 or 10 µM BD1047, as indicated. (K and L) Effects of siσ1R or control plasmid and pretreatment with σ1R ligands on the Ca 2+ signals evoked by 5 µM ionomycin in Ca 2+ -free HBS. Typical traces (K) and summary results (L) are shown. Legends for L are the same as J. All summary results show mean ± SEM. n = 3. *, P

Techniques Used: Transfection, Plasmid Preparation

Inhibition of SOCE by σ1R. (A) Ca 2+ signals recorded from populations of fluo 4–loaded HEK cells transiently transfected with Orai1 E106Q , STIM1 and Orai1, or mock transfected (control). Cells were stimulated with 5 µM thapsigargin in Ca 2+ -free HBS before restoration of extracellular Ca 2+ (final free [Ca 2+ ], 4 mM). Results show mean responses from six replicates. (B) Summary results ( n = 3) show peak increases in [Ca 2+ ] c evoked by thapsigargin (Ca 2+ release) and Ca 2+ restoration (SOCE). (C) Typical immunoblot of σ1R, STIM1, Orai1, and β-actin from 20 µg of solubilized protein from wild-type (WT) HEK and HEK-σ1R cells. (D) Ca 2+ signals evoked by thapsigargin in Ca 2+ -free HBS and after restoration of extracellular Ca 2+ to wild-type and HEK-σ1R cells. (E) Summary shows responses to thapsigargin (Ca 2+ release) and SOCE detected after restoring Ca 2+ 10 or 20 min after thapsigargin ( n = 6). (F) Responses from single fura 2–loaded HEK cells show fluorescence ratios (F 340 /F 380 ) after stimulation with 5 µM thapsigargin and restoration of 4 mM extracellular Ca 2+ . n = 3, each with ∼45 cells. (G) Ca 2+ contents of the intracellular stores determined by measuring [Ca 2+ ] c after addition of 5 µM ionomycin in Ca 2+ -free HBS before or 10 min after treatment with thapsigargin. (H) Summary results ( n = 6). (I) Ca 2+ release and SOCE evoked by 100 µM carbachol and 100 µM ATP. (J) Summary results ( n = 6). *, P
Figure Legend Snippet: Inhibition of SOCE by σ1R. (A) Ca 2+ signals recorded from populations of fluo 4–loaded HEK cells transiently transfected with Orai1 E106Q , STIM1 and Orai1, or mock transfected (control). Cells were stimulated with 5 µM thapsigargin in Ca 2+ -free HBS before restoration of extracellular Ca 2+ (final free [Ca 2+ ], 4 mM). Results show mean responses from six replicates. (B) Summary results ( n = 3) show peak increases in [Ca 2+ ] c evoked by thapsigargin (Ca 2+ release) and Ca 2+ restoration (SOCE). (C) Typical immunoblot of σ1R, STIM1, Orai1, and β-actin from 20 µg of solubilized protein from wild-type (WT) HEK and HEK-σ1R cells. (D) Ca 2+ signals evoked by thapsigargin in Ca 2+ -free HBS and after restoration of extracellular Ca 2+ to wild-type and HEK-σ1R cells. (E) Summary shows responses to thapsigargin (Ca 2+ release) and SOCE detected after restoring Ca 2+ 10 or 20 min after thapsigargin ( n = 6). (F) Responses from single fura 2–loaded HEK cells show fluorescence ratios (F 340 /F 380 ) after stimulation with 5 µM thapsigargin and restoration of 4 mM extracellular Ca 2+ . n = 3, each with ∼45 cells. (G) Ca 2+ contents of the intracellular stores determined by measuring [Ca 2+ ] c after addition of 5 µM ionomycin in Ca 2+ -free HBS before or 10 min after treatment with thapsigargin. (H) Summary results ( n = 6). (I) Ca 2+ release and SOCE evoked by 100 µM carbachol and 100 µM ATP. (J) Summary results ( n = 6). *, P

Techniques Used: Inhibition, Transfection, Fluorescence

STIM1, Orai1, and σ1R interact within a macromolecular complex at the PM. (A) HEK cells expressing σ1R-FLAG alone or with Orai1-Myc or Orai1-Myc and HA-STIM1 were treated with thapsigargin (5 µM for 30 min in Ca 2+ -free HBS), and then the cell surface was biotinylated. The representative immunoblot shows the inputs and the proteins detected after purification with avidin beads. Input lanes were loaded with 10 µl of the 500-µl sample, and surface biotinylation lanes were loaded with 10 µl of the 50-µl eluate. (B) Summary shows the amounts of σ1R-FLAG detected in the avidin pull-downs (normalized to cells expressing only σ1R-FLAG). (C) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were cell surface biotinylated before sequential purification by elution from avidin-agarose with biotin and then from anti-Myc­–agarose with Myc peptide. The immunoblot (anti-HA, anti-FLAG, anti-Myc, and anti–β-actin) shows the input and the two eluates. Input lanes were loaded with 10 µl of the 500-µl sample and elution lanes with 10 µl of the 50-µl eluate. (D) Summary shows the amounts of HA-STIM1 detected in the avidin (biotin elution) and anti-Myc pull-downs (normalized to Orai1-Myc pull-down in each condition). (E) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were immunoprecipitated (IP) with anti-HA antibody. (F) Peak [Ca 2+ ] c signals evoked by SOCE were recorded from HEK or HEK-σ1R cells after treatment with thapsigargin (5 µM in Ca 2+ -free HBS for 10 min) and then restoration of 4 mM extracellular Ca 2+ . The effects of transiently overexpressing STIM1 or Orai1 are shown. WT, wild type. (G) The Ca 2+ contents of the intracellular stores of the same cells were measured by recording peak increases in [Ca 2+ ] c from cells exposed to ionomycin (5 µM in Ca 2+ -free HBS). Results (B, D, F, and G) are mean ± SEM. n = 3. *, P
Figure Legend Snippet: STIM1, Orai1, and σ1R interact within a macromolecular complex at the PM. (A) HEK cells expressing σ1R-FLAG alone or with Orai1-Myc or Orai1-Myc and HA-STIM1 were treated with thapsigargin (5 µM for 30 min in Ca 2+ -free HBS), and then the cell surface was biotinylated. The representative immunoblot shows the inputs and the proteins detected after purification with avidin beads. Input lanes were loaded with 10 µl of the 500-µl sample, and surface biotinylation lanes were loaded with 10 µl of the 50-µl eluate. (B) Summary shows the amounts of σ1R-FLAG detected in the avidin pull-downs (normalized to cells expressing only σ1R-FLAG). (C) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were cell surface biotinylated before sequential purification by elution from avidin-agarose with biotin and then from anti-Myc­–agarose with Myc peptide. The immunoblot (anti-HA, anti-FLAG, anti-Myc, and anti–β-actin) shows the input and the two eluates. Input lanes were loaded with 10 µl of the 500-µl sample and elution lanes with 10 µl of the 50-µl eluate. (D) Summary shows the amounts of HA-STIM1 detected in the avidin (biotin elution) and anti-Myc pull-downs (normalized to Orai1-Myc pull-down in each condition). (E) HEK cells expressing Orai1-Myc and HA-STIM1 with or without σ1R-FLAG were immunoprecipitated (IP) with anti-HA antibody. (F) Peak [Ca 2+ ] c signals evoked by SOCE were recorded from HEK or HEK-σ1R cells after treatment with thapsigargin (5 µM in Ca 2+ -free HBS for 10 min) and then restoration of 4 mM extracellular Ca 2+ . The effects of transiently overexpressing STIM1 or Orai1 are shown. WT, wild type. (G) The Ca 2+ contents of the intracellular stores of the same cells were measured by recording peak increases in [Ca 2+ ] c from cells exposed to ionomycin (5 µM in Ca 2+ -free HBS). Results (B, D, F, and G) are mean ± SEM. n = 3. *, P

Techniques Used: Expressing, Purification, Avidin-Biotin Assay, Immunoprecipitation

14) Product Images from "Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells"

Article Title: Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12239

PGE 2 inhibits histamine-evoked Ca 2+ release. (A) Ca 2+ signals evoked by histamine (100 μM, bar) alone or with PGE 2 (10 μM, added 5 min before and then with histamine). Results, means ± SEM from three wells on a single plate, are typical of results from four independent plates. (B) Effect of PGE 2 (10 μM) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± SEM from seven independent plates, each with one to three wells. (C) Effect of PGE 2 on the sustained Ca 2+ signals evoked by histamine. Results are means ± SEM from 11 independent plates, each with one to three wells. (D) Effect of the indicated concentrations of PGE 2 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (3 μM). Results are means ± SEM from 15 independent plates, with one to three wells analysed from each. (B–D) Ct denotes control. Similar results from ASMC isolated from different patients are shown in Supporting Information Figure S1 . (E) Effects of pretreatment with PGE 2 (10 μM, 5 min) on the peak Ca 2+ signals evoked by subsequent addition of thapsigargin (1 μM), cyclopiazonic acid (10 μM) or ionomycin (1 μM) to ASMC in Ca 2+ -free HBS. Results (as percentages of the responses obtained without PGE 2 ) are means ± SEM from three independent plates, with seven wells analysed on each.
Figure Legend Snippet: PGE 2 inhibits histamine-evoked Ca 2+ release. (A) Ca 2+ signals evoked by histamine (100 μM, bar) alone or with PGE 2 (10 μM, added 5 min before and then with histamine). Results, means ± SEM from three wells on a single plate, are typical of results from four independent plates. (B) Effect of PGE 2 (10 μM) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± SEM from seven independent plates, each with one to three wells. (C) Effect of PGE 2 on the sustained Ca 2+ signals evoked by histamine. Results are means ± SEM from 11 independent plates, each with one to three wells. (D) Effect of the indicated concentrations of PGE 2 (added 5 min before histamine) on the peak increase in [Ca 2+ ] i evoked by histamine (3 μM). Results are means ± SEM from 15 independent plates, with one to three wells analysed from each. (B–D) Ct denotes control. Similar results from ASMC isolated from different patients are shown in Supporting Information Figure S1 . (E) Effects of pretreatment with PGE 2 (10 μM, 5 min) on the peak Ca 2+ signals evoked by subsequent addition of thapsigargin (1 μM), cyclopiazonic acid (10 μM) or ionomycin (1 μM) to ASMC in Ca 2+ -free HBS. Results (as percentages of the responses obtained without PGE 2 ) are means ± SEM from three independent plates, with seven wells analysed on each.

Techniques Used: Isolation

15) Product Images from "Cyclic AMP-induced Chromatin Changes Support the NFATc-mediated Recruitment of GATA-3 to the Interleukin 5 Promoter *"

Article Title: Cyclic AMP-induced Chromatin Changes Support the NFATc-mediated Recruitment of GATA-3 to the Interleukin 5 Promoter *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M805929200

Mapping of inducible DNase I hypersensitive sites within the murine il-5 locus . A and B , EL-4 cells were either left uninduced or treated for 6 h by 8-CTP-cAMP (cAMP), TPA, and ionomycin (T+I), or T+I and 8-CTP-cAMP (T+I+cAMP) as indicated. Nuclei
Figure Legend Snippet: Mapping of inducible DNase I hypersensitive sites within the murine il-5 locus . A and B , EL-4 cells were either left uninduced or treated for 6 h by 8-CTP-cAMP (cAMP), TPA, and ionomycin (T+I), or T+I and 8-CTP-cAMP (T+I+cAMP) as indicated. Nuclei

Techniques Used:

16) Product Images from "TCR-engineered T cells: A model of inducible TCR expression to dissect the interrelationship between two TCRs"

Article Title: TCR-engineered T cells: A model of inducible TCR expression to dissect the interrelationship between two TCRs

Journal: European Journal of Immunology

doi: 10.1002/eji.201343591

Inducible TCR expression in single TCR-Tg T cells. T-cell clones expressing P14 ind or OT-I ind TCR were seeded at a density of 4 × 10 5 /mL and incubated for 24 h with different concentrations of Dox. TCR expression was analyzed by flow cytometry and living cells discriminated in FSC/SSC dot plot. Dot plot of (A) P14 ind or (B) OT-I ind living cells stained for TCR chains. (C) Percentages or (D) MFI of TCRvα and TCRvβ chain double-positive cells as a function of Dox concentration. (E) Percentage of TCR-positive cells as a function of incubation time in presence of Dox (1 μg/mL). (C–E) Data are shown as mean ± SD ( n = 3–6) and are pooled from at least three independent experiments. (F) Co-cultivation of Dox-induced TCR-expressing 58 T cells with irradiated C57BL/6 splenocytes loaded with 10 μM peptide (gp 33–41 for P14 TCR and OVA 257–264 for OT-I TCR). As a control no peptide (w/o) or an unspecific stimulus with PMA and ionomycin (max) was added. IL-2 amount was determined by ELISA. Bars indicate mean of duplicates and mean deviation. One representative experiment of two is shown.
Figure Legend Snippet: Inducible TCR expression in single TCR-Tg T cells. T-cell clones expressing P14 ind or OT-I ind TCR were seeded at a density of 4 × 10 5 /mL and incubated for 24 h with different concentrations of Dox. TCR expression was analyzed by flow cytometry and living cells discriminated in FSC/SSC dot plot. Dot plot of (A) P14 ind or (B) OT-I ind living cells stained for TCR chains. (C) Percentages or (D) MFI of TCRvα and TCRvβ chain double-positive cells as a function of Dox concentration. (E) Percentage of TCR-positive cells as a function of incubation time in presence of Dox (1 μg/mL). (C–E) Data are shown as mean ± SD ( n = 3–6) and are pooled from at least three independent experiments. (F) Co-cultivation of Dox-induced TCR-expressing 58 T cells with irradiated C57BL/6 splenocytes loaded with 10 μM peptide (gp 33–41 for P14 TCR and OVA 257–264 for OT-I TCR). As a control no peptide (w/o) or an unspecific stimulus with PMA and ionomycin (max) was added. IL-2 amount was determined by ELISA. Bars indicate mean of duplicates and mean deviation. One representative experiment of two is shown.

Techniques Used: Expressing, Clone Assay, Incubation, Flow Cytometry, Cytometry, Staining, Concentration Assay, Irradiation, Enzyme-linked Immunosorbent Assay

17) Product Images from "Prostaglandin E2 Inhibits Histamine-Evoked Ca2+"

Article Title: Prostaglandin E2 Inhibits Histamine-Evoked Ca2+

Journal: Molecular Pharmacology

doi: 10.1124/mol.117.109249

Cyclic AMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 . (A) Effect of 8-Br-cAMP (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± S.E.M. from at least three experiments with one to three wells in each. (B) 8-Br-cAMP (10 mM, 20 minutes) had no effect on the Ca 2+ content of the intracellular stores as revealed by the increases in [Ca 2+ ] i evoked by addition of thapsigargin (1 μ M) or ionomycin (1 μ Μ) in Ca 2+ -free HBS. Results (percentages of responses without 8-Br-cAMP) are means ± S.E.M. from five experiments with three to four wells analyzed in each. (C) Effect of the indicated cyclic nucleotides (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by histamine (3 μ M). Results are means ± S.E.M. from three to five experiments with two to three wells in each. (D) Effect of NKH 477 (100 μ M, 5 minutes), forskolin (100 μ M, 5 minutes), 8-Br-cAMP (10 mM, 20 minutes), PGE 2 (10 μ Μ, 5 minutes), 8-Br-cGMP (10 mM, 20 minutes), R p-cAMPS (10 mM, 20 minutes), or 8-pCPT-2 ′ -O-Me-cAMP (10 mM, 20 minutes) alone or in combination on the peak Ca 2+ signals evoked by histamine (1 mM). Results (as percentages of the response to histamine alone) are means ± S.E.M. from three experiments with two to three wells in each. Results for R p-cAMPS are from a single experiment with three replicates, limited by the availability of this expensive analog. (E) Effects of the EPAC antagonists, ESI-09 and HJC0197 (10 μ M, 20 minutes), on the Ca 2+ signals evoked by histamine (3 μ M) added 5 minutes before and then during treatment with the indicated concentrations of PGE 2 . Results are expressed as percentages of the paired response to histamine alone (means ± S.E.M., n = 3–5; n = 2 for the antagonists with 1 and 3 nM PGE 2 , where error bars show ranges). (F) The results establish that cAMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 .
Figure Legend Snippet: Cyclic AMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 . (A) Effect of 8-Br-cAMP (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by the indicated concentrations of histamine. Results are means ± S.E.M. from at least three experiments with one to three wells in each. (B) 8-Br-cAMP (10 mM, 20 minutes) had no effect on the Ca 2+ content of the intracellular stores as revealed by the increases in [Ca 2+ ] i evoked by addition of thapsigargin (1 μ M) or ionomycin (1 μ Μ) in Ca 2+ -free HBS. Results (percentages of responses without 8-Br-cAMP) are means ± S.E.M. from five experiments with three to four wells analyzed in each. (C) Effect of the indicated cyclic nucleotides (added 20 minutes before histamine) on the peak Ca 2+ signals evoked by histamine (3 μ M). Results are means ± S.E.M. from three to five experiments with two to three wells in each. (D) Effect of NKH 477 (100 μ M, 5 minutes), forskolin (100 μ M, 5 minutes), 8-Br-cAMP (10 mM, 20 minutes), PGE 2 (10 μ Μ, 5 minutes), 8-Br-cGMP (10 mM, 20 minutes), R p-cAMPS (10 mM, 20 minutes), or 8-pCPT-2 ′ -O-Me-cAMP (10 mM, 20 minutes) alone or in combination on the peak Ca 2+ signals evoked by histamine (1 mM). Results (as percentages of the response to histamine alone) are means ± S.E.M. from three experiments with two to three wells in each. Results for R p-cAMPS are from a single experiment with three replicates, limited by the availability of this expensive analog. (E) Effects of the EPAC antagonists, ESI-09 and HJC0197 (10 μ M, 20 minutes), on the Ca 2+ signals evoked by histamine (3 μ M) added 5 minutes before and then during treatment with the indicated concentrations of PGE 2 . Results are expressed as percentages of the paired response to histamine alone (means ± S.E.M., n = 3–5; n = 2 for the antagonists with 1 and 3 nM PGE 2 , where error bars show ranges). (F) The results establish that cAMP mediates inhibition of histamine-evoked Ca 2+ signals by PGE 2 .

Techniques Used: Inhibition

18) Product Images from "VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling"

Article Title: VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.118.311118

The generation of NRP1 (neuropilin-1) cytoplasmic fragments in human umbilical vein endothelial cells (HUVECs) is mediated by metalloproteinase activity. A , Increasing concentrations of marimastat, a broad-spectrum metalloproteinase inhibitor, reduces the expression of the 10 and 15 kDa fragments detected by antibody specific for the NRP1 cytoplasmic domain (C-term) in a dose-dependent manner. B , The dose-response curve for the effect of marimastat on generation of the 10 kDa NRP1 cytoplasmic fragment indicates an IC 50 of 10 μmol\L, similar to reported values for marimastat. C , HUVECs, either infected with Ad.NRP1WT or uninfected (UI) were treated for 24 h with Phorbol ester (PMA), or Ionomycin (IM), or vehicle (DMSO) control (VC), at the indicated concentrations, and cell lysates were then prepared and immunoblotted as shown.
Figure Legend Snippet: The generation of NRP1 (neuropilin-1) cytoplasmic fragments in human umbilical vein endothelial cells (HUVECs) is mediated by metalloproteinase activity. A , Increasing concentrations of marimastat, a broad-spectrum metalloproteinase inhibitor, reduces the expression of the 10 and 15 kDa fragments detected by antibody specific for the NRP1 cytoplasmic domain (C-term) in a dose-dependent manner. B , The dose-response curve for the effect of marimastat on generation of the 10 kDa NRP1 cytoplasmic fragment indicates an IC 50 of 10 μmol\L, similar to reported values for marimastat. C , HUVECs, either infected with Ad.NRP1WT or uninfected (UI) were treated for 24 h with Phorbol ester (PMA), or Ionomycin (IM), or vehicle (DMSO) control (VC), at the indicated concentrations, and cell lysates were then prepared and immunoblotted as shown.

Techniques Used: Activity Assay, Expressing, Infection

19) Product Images from "VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling"

Article Title: VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.118.311118

The generation of NRP1 (neuropilin-1) cytoplasmic fragments in human umbilical vein endothelial cells (HUVECs) is mediated by metalloproteinase activity. A , Increasing concentrations of marimastat, a broad-spectrum metalloproteinase inhibitor, reduces the expression of the 10 and 15 kDa fragments detected by antibody specific for the NRP1 cytoplasmic domain (C-term) in a dose-dependent manner. B , The dose-response curve for the effect of marimastat on generation of the 10 kDa NRP1 cytoplasmic fragment indicates an IC 50 of 10 μmol\L, similar to reported values for marimastat. C , HUVECs, either infected with Ad.NRP1WT or uninfected (UI) were treated for 24 h with Phorbol ester (PMA), or Ionomycin (IM), or vehicle (DMSO) control (VC), at the indicated concentrations, and cell lysates were then prepared and immunoblotted as shown.
Figure Legend Snippet: The generation of NRP1 (neuropilin-1) cytoplasmic fragments in human umbilical vein endothelial cells (HUVECs) is mediated by metalloproteinase activity. A , Increasing concentrations of marimastat, a broad-spectrum metalloproteinase inhibitor, reduces the expression of the 10 and 15 kDa fragments detected by antibody specific for the NRP1 cytoplasmic domain (C-term) in a dose-dependent manner. B , The dose-response curve for the effect of marimastat on generation of the 10 kDa NRP1 cytoplasmic fragment indicates an IC 50 of 10 μmol\L, similar to reported values for marimastat. C , HUVECs, either infected with Ad.NRP1WT or uninfected (UI) were treated for 24 h with Phorbol ester (PMA), or Ionomycin (IM), or vehicle (DMSO) control (VC), at the indicated concentrations, and cell lysates were then prepared and immunoblotted as shown.

Techniques Used: Activity Assay, Expressing, Infection

20) Product Images from "Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita"

Article Title: Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083631

CD4 T cells specific for various antigens detected after mCOL7c-GST immunization. Draining lymph node cells were harvested 5 weeks after immunization and restimulated with no antigen, OVA, mCOL7c-HIS, GST or mCOL7c-GST, as indicated. T cells specific for the respective antigens were identified by electronic gating on CD4+ cells and the rapid up-regulation of CD154 [32] . A, representative flow cytometric analysis of cells stimulated with various antigens. Restimulation with PMA/Ionomycin was used as a positive control. B, Frequencies of antigen-specific cells obtained from draining lymph nodes 5 weeks after mCOL7c-GST immunization are shown (n = 6). Similar frequencies were determined at week 3 after immunization (data not shown). Data are representative for more than three independent experiments.
Figure Legend Snippet: CD4 T cells specific for various antigens detected after mCOL7c-GST immunization. Draining lymph node cells were harvested 5 weeks after immunization and restimulated with no antigen, OVA, mCOL7c-HIS, GST or mCOL7c-GST, as indicated. T cells specific for the respective antigens were identified by electronic gating on CD4+ cells and the rapid up-regulation of CD154 [32] . A, representative flow cytometric analysis of cells stimulated with various antigens. Restimulation with PMA/Ionomycin was used as a positive control. B, Frequencies of antigen-specific cells obtained from draining lymph nodes 5 weeks after mCOL7c-GST immunization are shown (n = 6). Similar frequencies were determined at week 3 after immunization (data not shown). Data are representative for more than three independent experiments.

Techniques Used: Flow Cytometry, Positive Control

mCOL7c-GST specific CD4 T cells detected in spleen and draining lymph nodes after mCOL7c-GST immunization. Draining lymph node and spleen cells were harvested 7 weeks after immunization and restimulated with OVA (negative control), mCOL7c-GST or PMA and Ionomycin (positive control). A, representative flow cytometric analysis of restimulated CD 4 T cells. Cells were pre-gated according to CD4 expression. B, Frequencies of mCOL7c-specific cells obtained from draining lymph nodes and spleens 7 weeks after mCOL7c-GST immunization are shown (n = 8). Data are representative for more than three independent experiments.
Figure Legend Snippet: mCOL7c-GST specific CD4 T cells detected in spleen and draining lymph nodes after mCOL7c-GST immunization. Draining lymph node and spleen cells were harvested 7 weeks after immunization and restimulated with OVA (negative control), mCOL7c-GST or PMA and Ionomycin (positive control). A, representative flow cytometric analysis of restimulated CD 4 T cells. Cells were pre-gated according to CD4 expression. B, Frequencies of mCOL7c-specific cells obtained from draining lymph nodes and spleens 7 weeks after mCOL7c-GST immunization are shown (n = 8). Data are representative for more than three independent experiments.

Techniques Used: Negative Control, Positive Control, Flow Cytometry, Expressing

21) Product Images from "Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line"

Article Title: Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line

Journal: International Journal of Cancer

doi: 10.1002/ijc.20779

D4 can kill transfectants expressing the full‐length E7 of HPV types 16 and 52.The CTL D4 was assessed for its ability to kill transfectants expressing full length E7 of HPV types 16, 31, 45 and 52. C33A cells were transfected using the liposomal transfection reagent DOTAP and selected by growing in media containing 2.5 μg/ml blasticidin. The number of antigen specific T cells was analyzed by IFNγ ELISPOT. Responder cells were incubated with the following stimulators: C33A; C33A HPV16, C33A transfected in the laboratory to express HPV16 (C33AHPV16A), HPV31, HPV45 and HPV52. The ELISPOT was developed after 18 hr and cells were mixed at ratio of 1:1 with a total number of 20,000 responders/well. *Transfectants that stimulated CTL to produce a significantly higher amount of IFNγ compared to wild‐type C33A cells incubated with D4. Under the same culture conditions, incubation of D4 with the HPV16 E7 11–20 peptide at 10 μg/ml produced 404 spots and with a positive control cocktail containing PHA, PMA, concavalin A and ionomycin more than 500 spots was seen (data not shown).
Figure Legend Snippet: D4 can kill transfectants expressing the full‐length E7 of HPV types 16 and 52.The CTL D4 was assessed for its ability to kill transfectants expressing full length E7 of HPV types 16, 31, 45 and 52. C33A cells were transfected using the liposomal transfection reagent DOTAP and selected by growing in media containing 2.5 μg/ml blasticidin. The number of antigen specific T cells was analyzed by IFNγ ELISPOT. Responder cells were incubated with the following stimulators: C33A; C33A HPV16, C33A transfected in the laboratory to express HPV16 (C33AHPV16A), HPV31, HPV45 and HPV52. The ELISPOT was developed after 18 hr and cells were mixed at ratio of 1:1 with a total number of 20,000 responders/well. *Transfectants that stimulated CTL to produce a significantly higher amount of IFNγ compared to wild‐type C33A cells incubated with D4. Under the same culture conditions, incubation of D4 with the HPV16 E7 11–20 peptide at 10 μg/ml produced 404 spots and with a positive control cocktail containing PHA, PMA, concavalin A and ionomycin more than 500 spots was seen (data not shown).

Techniques Used: Expressing, Transfection, Enzyme-linked Immunospot, Incubation, Produced, Positive Control

Related Articles

Flow Cytometry:

Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion
Article Snippet: .. Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany). .. IFN-γ ELISPOT and IFN-γ/Granzyme B/Perforin ELISA CD8+ cells were purified from the MART-1aa26–35*A27L activated and expanded T cell population using positive immunomagnetic cell sorting (MACS-system, Miltenyi Biotec).

In Vitro:

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection
Article Snippet: .. For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C. ..

Positive Control:

Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion
Article Snippet: .. Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany). .. IFN-γ ELISPOT and IFN-γ/Granzyme B/Perforin ELISA CD8+ cells were purified from the MART-1aa26–35*A27L activated and expanded T cell population using positive immunomagnetic cell sorting (MACS-system, Miltenyi Biotec).

Cytometry:

Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion
Article Snippet: .. Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany). .. IFN-γ ELISPOT and IFN-γ/Granzyme B/Perforin ELISA CD8+ cells were purified from the MART-1aa26–35*A27L activated and expanded T cell population using positive immunomagnetic cell sorting (MACS-system, Miltenyi Biotec).

Infection:

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection
Article Snippet: .. For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C. ..

Mouse Assay:

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection
Article Snippet: .. For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C. ..

Concentration Assay:

Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion
Article Snippet: .. Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany). .. IFN-γ ELISPOT and IFN-γ/Granzyme B/Perforin ELISA CD8+ cells were purified from the MART-1aa26–35*A27L activated and expanded T cell population using positive immunomagnetic cell sorting (MACS-system, Miltenyi Biotec).

Incubation:

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection
Article Snippet: .. For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C. ..

other:

Article Title: Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation
Article Snippet: Ionomycin was purchased from Merck Millipore.

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Activity Assay:

Article Title: Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion
Article Snippet: .. To stimulate NFATC1 activity and nuclear translocation, cells were treated for 7 h with 15 ng/ml of 12–0-tetradecanoyl-phorbol-13-asetate (TPA; Sigma-Aldrich, St. Louis, MO, USA) in DMSO and 1 μM ionomycin (IM; Merck KGaA, Darmstadt, Germany) in EtOH. .. To inhibit PIM kinase activity, cells were treated for 24 h with 10 μM DHPCC-9 in 0,1% DMSO.

Staining:

Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection
Article Snippet: .. For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C. ..

Translocation Assay:

Article Title: Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion
Article Snippet: .. To stimulate NFATC1 activity and nuclear translocation, cells were treated for 7 h with 15 ng/ml of 12–0-tetradecanoyl-phorbol-13-asetate (TPA; Sigma-Aldrich, St. Louis, MO, USA) in DMSO and 1 μM ionomycin (IM; Merck KGaA, Darmstadt, Germany) in EtOH. .. To inhibit PIM kinase activity, cells were treated for 24 h with 10 μM DHPCC-9 in 0,1% DMSO.

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    Merck KGaA ionomycin
    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore <t>ionomycin</t> was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.
    Ionomycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Journal: PLoS ONE

    Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

    doi: 10.1371/journal.pone.0063521

    Figure Lengend Snippet: Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Article Snippet: Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany).

    Techniques: Chemotaxis Assay, Purification, Migration, Flow Cytometry, Cytometry, Negative Control, Positive Control

    Accelerated lipid oxidation is essential for SSZ-induced NETosis. ( a – d ) Mouse neutrophils were stimulated with various concentrations of PMA ( a , b ) or ionomycin ( c , d ) in the presence or absence of 1 mM SSZ for 1 h. C11-Bodipy 581/591 was then added. ( a , c ) The accumulation of lipid oxidation was analyzed using flow cytometry. ( b , d ) Average mean fluorescent intensity (MFI) of C11-Bodipy analysis with s.d. of triplicated samples are shown. * P

    Journal: Scientific Reports

    Article Title: Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation

    doi: 10.1038/s41598-017-15668-z

    Figure Lengend Snippet: Accelerated lipid oxidation is essential for SSZ-induced NETosis. ( a – d ) Mouse neutrophils were stimulated with various concentrations of PMA ( a , b ) or ionomycin ( c , d ) in the presence or absence of 1 mM SSZ for 1 h. C11-Bodipy 581/591 was then added. ( a , c ) The accumulation of lipid oxidation was analyzed using flow cytometry. ( b , d ) Average mean fluorescent intensity (MFI) of C11-Bodipy analysis with s.d. of triplicated samples are shown. * P

    Article Snippet: Ionomycin was purchased from Merck Millipore.

    Techniques: Flow Cytometry, Cytometry

    Claspin mRNA is regulated independently of the cell cycle but Claspin is responsive to NF-κB activation. ( A ) U2OS cells were depleted of IKKα, IKKβ, Claspin or Cyclin D1 using siRNA. WCLs were subjected to western blot analysis for the levels of the indicated proteins. ( B ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of IKKα, IKKβ, Claspin or Cyclin D1 by siRNA. ANOVA t -tests were performed on the means, and the P -values were calculated. ** P ⩽0.01 and *** P ⩽0.001. ( C ) U2OS cells were synchronized at G1/S using a double-thymidine block. Thymidine was subsequently washed and cells were released for the indicated times. Cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared from these cells and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( D ) U2OS cells were synchronized at prometaphase using nocodazole. Nocodazole was subsequently washed and cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( E ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of β-TrCP by siRNA. ( F ) U2OS cells were treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h. One hour prior to harvesting, cells were treated with UV (40 J/m 2 ) as indicated. WCLs were prepared and Claspin levels were analysed by western blot.

    Journal: The EMBO Journal

    Article Title: IKK and NF-?B-mediated regulation of Claspin impacts on ATR checkpoint function

    doi: 10.1038/emboj.2010.171

    Figure Lengend Snippet: Claspin mRNA is regulated independently of the cell cycle but Claspin is responsive to NF-κB activation. ( A ) U2OS cells were depleted of IKKα, IKKβ, Claspin or Cyclin D1 using siRNA. WCLs were subjected to western blot analysis for the levels of the indicated proteins. ( B ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of IKKα, IKKβ, Claspin or Cyclin D1 by siRNA. ANOVA t -tests were performed on the means, and the P -values were calculated. ** P ⩽0.01 and *** P ⩽0.001. ( C ) U2OS cells were synchronized at G1/S using a double-thymidine block. Thymidine was subsequently washed and cells were released for the indicated times. Cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared from these cells and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( D ) U2OS cells were synchronized at prometaphase using nocodazole. Nocodazole was subsequently washed and cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. ( E ) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of β-TrCP by siRNA. ( F ) U2OS cells were treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h. One hour prior to harvesting, cells were treated with UV (40 J/m 2 ) as indicated. WCLs were prepared and Claspin levels were analysed by western blot.

    Article Snippet: PMA (Sigma) and Ionomycin (Merck Biosciences) were dissolved in DMSO.

    Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Blocking Assay, Flow Cytometry, Cytometry

    Loss or inhibition of IKKβ disrupts Chk1 phosphorylation in response to UV. ( A ) U2OS cells were depleted of IKKα, IKKβ, IKKγ or Claspin with siRNA. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( B ) U2OS cell were pretreated with the IKK inhibitor, Bay 11-7082, for 24 h, as indicated. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( C ) U2OS cells were depleted of the NF-κB transcription factors RelA, RelB, c-Rel, p50 or p52 with siRNA. Cells were treated with UV as in ( A ) and WCLs prepared analysed by western blot using the indicated antibodies. ( D ) U2OS cells were transfected with control or constitutive active IKKβ (left panel) or treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h (right panel) prior to treatment with UV (40 J/m 2 ) as indicated. WCLs were prepared and analysed by western blot using the indicated antibodies. ( E ) U2OS cells were transfected with a non-targeting siRNA or a siRNA to deplete IKKβ. c-Rel was then re-introduced into these cells by transient transfection, and cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies. ( F ) U2OS cells were co-transfected with a non-targeting siRNA or a siRNA to deplete IKKβ, along with a DNA construct to re-introduce Claspin. Cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies.

    Journal: The EMBO Journal

    Article Title: IKK and NF-?B-mediated regulation of Claspin impacts on ATR checkpoint function

    doi: 10.1038/emboj.2010.171

    Figure Lengend Snippet: Loss or inhibition of IKKβ disrupts Chk1 phosphorylation in response to UV. ( A ) U2OS cells were depleted of IKKα, IKKβ, IKKγ or Claspin with siRNA. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( B ) U2OS cell were pretreated with the IKK inhibitor, Bay 11-7082, for 24 h, as indicated. Cells were treated with UV (40 J/m 2 ) as indicated, and harvested 4 h later. WCLs were prepared and analysed by western blot using the indicated antibodies. ( C ) U2OS cells were depleted of the NF-κB transcription factors RelA, RelB, c-Rel, p50 or p52 with siRNA. Cells were treated with UV as in ( A ) and WCLs prepared analysed by western blot using the indicated antibodies. ( D ) U2OS cells were transfected with control or constitutive active IKKβ (left panel) or treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h (right panel) prior to treatment with UV (40 J/m 2 ) as indicated. WCLs were prepared and analysed by western blot using the indicated antibodies. ( E ) U2OS cells were transfected with a non-targeting siRNA or a siRNA to deplete IKKβ. c-Rel was then re-introduced into these cells by transient transfection, and cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies. ( F ) U2OS cells were co-transfected with a non-targeting siRNA or a siRNA to deplete IKKβ, along with a DNA construct to re-introduce Claspin. Cells were treated with UV (40 J/m 2 ) as indicated, and left to culture for a further 4 h. WCLs were prepared and analysed by western blot using the indicated antibodies.

    Article Snippet: PMA (Sigma) and Ionomycin (Merck Biosciences) were dissolved in DMSO.

    Techniques: Inhibition, Western Blot, Transfection, Construct, Introduce

    A129 mice 4-week-old survive to ZIKV PE243 and evoke a robust immune response. Three- and 4-week-old A129 mice were inoculated intravenously with 2 × 10 5 PFU of ZIKV strain PE243. Uninfected mice were used as control. a , b Body weight gain ( a ) and lethality ( b ) of 3- ( n = 9) or 4-week-old A129 mice ( n = 15) infected with ZIKV PE243 were monitored for up to 15 days postinfection. c ZIKV RNA copies in the spleen, kidney, liver, and brain determined by qRT-PCR at days 3, 5, and 7 postinfection of 3- ( n = 3–8) or 4-week-old mice ( n = 4–6). Results are expressed as RNA equivalent/copies and normalized by GAPDH. Flow cytometry were performed on splenocytes from uninfected ( n = 4–8) or 4-week-old mice at day 7 postinfection ( n = 4–15). For cytokines and cytotoxic factors detection, splenocytes were restimulated in vitro with PMA and ionomycin in the presence of brefeldin A during 4 h. d Representative dot plot and frequency of CD62L − CD44 + (effector) among CD8 + . e Representative counter plot and frequency of IFNγ, IL-2, CD107a, granzyme B (GrzmB), and perforin (Prfn) among CD8 + T cells. f Representative dot plot and frequency of CD62L - CD44 + (effector) among CD4 + . g Representative counter plot and frequency of I IFNγ, IL-4, and IL-17 among CD4 + T cells. h Representative counter plot and frequency of CD25 + Foxp3 + or CXCR5 + PD-1 + and CXCR5 + PD-1 + IFNγ + among CD4 + T cells. i Representative counter plot and frequency of germinal center B cells among B220 + CD138 − and plasma cells. Results are shown as mean in d – i or as mean ± standard deviation in a and c . Data are representative of two independent experiments in a , b , and c , data are presented as a pool of two or three independent experiments in d – i . Survival data were analyzed by log rank test. Data were analyzed by Student’s t test in c–i. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

    Journal: Nature Communications

    Article Title: Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection

    doi: 10.1038/s41467-018-05519-4

    Figure Lengend Snippet: A129 mice 4-week-old survive to ZIKV PE243 and evoke a robust immune response. Three- and 4-week-old A129 mice were inoculated intravenously with 2 × 10 5 PFU of ZIKV strain PE243. Uninfected mice were used as control. a , b Body weight gain ( a ) and lethality ( b ) of 3- ( n = 9) or 4-week-old A129 mice ( n = 15) infected with ZIKV PE243 were monitored for up to 15 days postinfection. c ZIKV RNA copies in the spleen, kidney, liver, and brain determined by qRT-PCR at days 3, 5, and 7 postinfection of 3- ( n = 3–8) or 4-week-old mice ( n = 4–6). Results are expressed as RNA equivalent/copies and normalized by GAPDH. Flow cytometry were performed on splenocytes from uninfected ( n = 4–8) or 4-week-old mice at day 7 postinfection ( n = 4–15). For cytokines and cytotoxic factors detection, splenocytes were restimulated in vitro with PMA and ionomycin in the presence of brefeldin A during 4 h. d Representative dot plot and frequency of CD62L − CD44 + (effector) among CD8 + . e Representative counter plot and frequency of IFNγ, IL-2, CD107a, granzyme B (GrzmB), and perforin (Prfn) among CD8 + T cells. f Representative dot plot and frequency of CD62L - CD44 + (effector) among CD4 + . g Representative counter plot and frequency of I IFNγ, IL-4, and IL-17 among CD4 + T cells. h Representative counter plot and frequency of CD25 + Foxp3 + or CXCR5 + PD-1 + and CXCR5 + PD-1 + IFNγ + among CD4 + T cells. i Representative counter plot and frequency of germinal center B cells among B220 + CD138 − and plasma cells. Results are shown as mean in d – i or as mean ± standard deviation in a and c . Data are representative of two independent experiments in a , b , and c , data are presented as a pool of two or three independent experiments in d – i . Survival data were analyzed by log rank test. Data were analyzed by Student’s t test in c–i. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

    Article Snippet: For unspecific stimulation, cells were incubated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL) (Sigma-Aldrich) and ionomycin (4 μg/mL) (Merck Milipore, Billerica, MA) in the presence of Brefeldin A X1 (eBioscience, San Diego, CA) at 37 °C and 5% CO2 , during 4 h. In order to evaluate ZIKV-specific responses, cells from infected mice were harvested at 5 × 105 cells/well in a 96-well plate and restimulated in vitro using UV-inactivated virus and maintained in supplemented RPMI medium (Lonza, Baltimore, MD) at 37 °C with 5% CO2 during 72 h. Brefeldin A X1 was added at the cultures on the last 8 h. After specific or unspecific in vitro stimulation, cells were stained with antibodies against surface markers conjugated with fluorochromes, during 30 min at 4 °C.

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR, Flow Cytometry, Cytometry, In Vitro, Standard Deviation