Structured Review

Enzo Biochem ionomycin
Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and <t>ionomycin</t> for subsequent 5 h. Th cells were identified
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1) Product Images from "Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells [S]"

Article Title: Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.P031260

Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and ionomycin for subsequent 5 h. Th cells were identified
Figure Legend Snippet: Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and ionomycin for subsequent 5 h. Th cells were identified

Techniques Used: Incubation

2) Product Images from "Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells"

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

Journal: Nature Communications

doi: 10.1038/s41467-019-09431-3

Shp-2 is largely dispensable for NK cell effector functions. a Graphs depict percentages of CD94 + , Ly49A + , Ly49G2 + , Ly49I + , Ly49D + , Ly49H + , and KLRG1 + splenic NK cells (gated as NK1.1 + CD3/CD19 − ) from Ncr1 Ki Ptpn11 wt/wt (white), Ncr1 Ki Ptpn11 fl/fl (dark gray), and B2m −/− (green) mice. b Graph and a representative cytometric plot illustrate the production of granzyme A and B by splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) after phorbol 12-myristate 13-acetate and ionomycin stimulation (PMA/Iono). c NK cells isolated from polyinosinic:polycytidylic acid (polyI:C)-treated Ncr1 Ki Ptpn11 wt/wt or Ncr1 Ki Ptpn11 fl/fl mice were plated with RMA, RMA-S, or RMA-H60 cells at the indicated ratios. The graph depicts percentage killing of target cells, as measured by quantifying PI − living target cells. d Naive splenocytes from Ncr1 Tg Ptpn11 fl/fl (light gray) mice or heterozygote littermate controls (white) were incubated with RMA cells or RMA-m157 cells (expression of m157 is illustrated in the graph on the left). Percentage of YFP + Ly49H + CD107α + NK cells in each group was determined by flow cytometry (illustrated in the graph on the right). a , b Results represent the mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl or B2m −/− ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) mice per genotype and are representative of at least three ( a ) and two ( b ) independent experiments. c , d Results represent the mean ± SD of n = 3 ( c ), n = 4 (for non-stimulated conditions) and n = 5 (for RMA conditions) ( d ) technical replicates and are representative of at least two ( c ) and three ( d ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file
Figure Legend Snippet: Shp-2 is largely dispensable for NK cell effector functions. a Graphs depict percentages of CD94 + , Ly49A + , Ly49G2 + , Ly49I + , Ly49D + , Ly49H + , and KLRG1 + splenic NK cells (gated as NK1.1 + CD3/CD19 − ) from Ncr1 Ki Ptpn11 wt/wt (white), Ncr1 Ki Ptpn11 fl/fl (dark gray), and B2m −/− (green) mice. b Graph and a representative cytometric plot illustrate the production of granzyme A and B by splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) after phorbol 12-myristate 13-acetate and ionomycin stimulation (PMA/Iono). c NK cells isolated from polyinosinic:polycytidylic acid (polyI:C)-treated Ncr1 Ki Ptpn11 wt/wt or Ncr1 Ki Ptpn11 fl/fl mice were plated with RMA, RMA-S, or RMA-H60 cells at the indicated ratios. The graph depicts percentage killing of target cells, as measured by quantifying PI − living target cells. d Naive splenocytes from Ncr1 Tg Ptpn11 fl/fl (light gray) mice or heterozygote littermate controls (white) were incubated with RMA cells or RMA-m157 cells (expression of m157 is illustrated in the graph on the left). Percentage of YFP + Ly49H + CD107α + NK cells in each group was determined by flow cytometry (illustrated in the graph on the right). a , b Results represent the mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl or B2m −/− ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) mice per genotype and are representative of at least three ( a ) and two ( b ) independent experiments. c , d Results represent the mean ± SD of n = 3 ( c ), n = 4 (for non-stimulated conditions) and n = 5 (for RMA conditions) ( d ) technical replicates and are representative of at least two ( c ) and three ( d ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Techniques Used: Mouse Assay, Isolation, Incubation, Expressing, Flow Cytometry, Cytometry

3) Product Images from "Increased Number of Tc17 and Correlation with Th17 Cells in Patients with Immune Thrombocytopenia"

Article Title: Increased Number of Tc17 and Correlation with Th17 Cells in Patients with Immune Thrombocytopenia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026522

The percentages of circulating Tc17 and Th17 cells in ITP patients and controls. Heparinized peripheral whole blood from all subjects was stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, then stained with labeled antibodies as described in the Design and Methods section. (a) Lymphocytes were gated by flow cytometry in Gate 1. (b,d,f) CD3+ T subsets were gated by flow cytometry; the plots in intern box Gate 2 represent CD3+ T cells. (c, e, g) The percentages of circulating Tc17 and Th17 cells from ITP patients and controls; the percentage of positive cells is shown in each panel.
Figure Legend Snippet: The percentages of circulating Tc17 and Th17 cells in ITP patients and controls. Heparinized peripheral whole blood from all subjects was stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, then stained with labeled antibodies as described in the Design and Methods section. (a) Lymphocytes were gated by flow cytometry in Gate 1. (b,d,f) CD3+ T subsets were gated by flow cytometry; the plots in intern box Gate 2 represent CD3+ T cells. (c, e, g) The percentages of circulating Tc17 and Th17 cells from ITP patients and controls; the percentage of positive cells is shown in each panel.

Techniques Used: Staining, Labeling, Flow Cytometry, Cytometry

4) Product Images from "Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK."

Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

Journal: FEBS letters

doi:

Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.
Figure Legend Snippet: Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.

Techniques Used: Activation Assay

Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.
Figure Legend Snippet: Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.

Techniques Used: Activation Assay, Incubation, Western Blot

5) Product Images from "Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome"

Article Title: Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051339

Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4 + IL-17 + ) cells from healthy controls, E-MDS and L-MDS patients. (E, F, G) Representative FACS dot plots of circulating Th1 (CD4 + IFNγ + ) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4 + IL-22 + IL-17 − IFNγ − ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant.
Figure Legend Snippet: Circulating percentages of Th17, Th1 and Th22 cells in representative healthy controls, E-MDS and L-MDS patients. Heparinized peripheral whole blood from 37 MDS(E-MDS, n = 17; L-MDS, n = 20)patients and 20 healthy PB donors were stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h, and then stained with labeled antibodies for FACS analysis. (A) Lymphocytes were gated by flow cytometry. (B, C, D) Representative FACS dot plots of circulating Th17 (CD4 + IL-17 + ) cells from healthy controls, E-MDS and L-MDS patients. (E, F, G) Representative FACS dot plots of circulating Th1 (CD4 + IFNγ + ) cells from healthy controls, E-MDS and L-MDS patients. (H, I, J) Representative FACS dot plots of circulating Th22 (CD4 + IL-22 + IL-17 − IFNγ − ) cells from healthy controls, E-MDS and L-MDS patients. Numbers in plots indicate relative percentages per quadrant.

Techniques Used: Staining, Labeling, FACS, Flow Cytometry, Cytometry

6) Product Images from "Increased Frequencies of Th22 Cells as well as Th17 Cells in the Peripheral Blood of Patients with Ankylosing Spondylitis and Rheumatoid Arthritis"

Article Title: Increased Frequencies of Th22 Cells as well as Th17 Cells in the Peripheral Blood of Patients with Ankylosing Spondylitis and Rheumatoid Arthritis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031000

Circulating Th22 cells and CD4 + IFNγ - IL17 + IL-22 + T cells are significantly increased in ankylosing spondylitis (AS) patients and rheumatoid arthritis (RA) patients compared with osteoarthritis (OA) patients and healthy controls. a, Representative flow cytometry dot plots example of each group. b, The percentages of circulating Th22 cells (left panel), Th1 cells (middle panel) and CD4 + IFNγ - IL17 + IL-22 + T cells (right panel) from AS, RA, OA patients and healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. (* = P
Figure Legend Snippet: Circulating Th22 cells and CD4 + IFNγ - IL17 + IL-22 + T cells are significantly increased in ankylosing spondylitis (AS) patients and rheumatoid arthritis (RA) patients compared with osteoarthritis (OA) patients and healthy controls. a, Representative flow cytometry dot plots example of each group. b, The percentages of circulating Th22 cells (left panel), Th1 cells (middle panel) and CD4 + IFNγ - IL17 + IL-22 + T cells (right panel) from AS, RA, OA patients and healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. (* = P

Techniques Used: Flow Cytometry, Cytometry

Circulating Th17 cells and Th17/Th1 cells are significantly increased in ankylosing spondylitis (AS) patients and rheumatoid arthritis (RA) patients compared with osteoarthritis (OA) patients and healthy controls. a, Representative flow cytometry dot plots example of each group. b, The percentages of circulating Th17 cells (left panel) and Th17/Th1 cells (right panel) from AS, RA, OA patients and healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h (* = P
Figure Legend Snippet: Circulating Th17 cells and Th17/Th1 cells are significantly increased in ankylosing spondylitis (AS) patients and rheumatoid arthritis (RA) patients compared with osteoarthritis (OA) patients and healthy controls. a, Representative flow cytometry dot plots example of each group. b, The percentages of circulating Th17 cells (left panel) and Th17/Th1 cells (right panel) from AS, RA, OA patients and healthy controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h (* = P

Techniques Used: Flow Cytometry, Cytometry

7) Product Images from "Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation"

Article Title: Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation

Journal: Nature communications

doi: 10.1038/ncomms4551

T cells require Bhlhe40 for normal cytokine production after immunization a , b , ELISPOT assays for the quantitation of cells secreting ( a ) IL-2, IFN-γ, and IL-17A or ( b ) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40 −/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c , e , DLN cells from immunized WT and Bhlhe40 −/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. ( c ) GM-CSF or ( e ) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. f , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. g , Frequencies of GM-CSF + and IL-17A + γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40 −/− mice (n=3 per group) as determined by ICS. h , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.
Figure Legend Snippet: T cells require Bhlhe40 for normal cytokine production after immunization a , b , ELISPOT assays for the quantitation of cells secreting ( a ) IL-2, IFN-γ, and IL-17A or ( b ) GM-CSF and IL-10 performed on DLN cells 7 days after immunization of WT and Bhlhe40 −/− mice. Data for IL-2, IFN-γ, IL-17A, and GM-CSF are combined from 3 independent experiments (n=9 mice per group). Data for IL-10 is from one representative experiment of two (n=4 mice per group). c , e , DLN cells from immunized WT and Bhlhe40 −/− mice (n=14 per group) were cultured with or without MOG(35-55) and with or without IL-1β, IL-23, and/or IL-12 as indicated. ( c ) GM-CSF or ( e ) IL-10 was measured in the supernatant at day 4. Data are combined from 5 independent experiments. Cells from all mice were not used in all conditions in each of the 4 experiments. d , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-1β for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. f , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with MOG(35-55) with or without IL-12 for 4 days, followed by ICS. Representative plots are gated on CD4 + T cells. g , Frequencies of GM-CSF + and IL-17A + γδ T cells in DLNs 7 days after immunization of WT and Bhlhe40 −/− mice (n=3 per group) as determined by ICS. h , DLN cells from immunized WT and Bhlhe40 −/− mice were cultured with or without MOG(35-55) and with or without IL-1β and/or IL-23 as indicated for 4 days. Cells were stimulated with PMA/ionomycin in the presence of brefeldin A for 4 hours and then analyzed for IL-17A and GM-CSF by intracellular staining (i.e. our normal ICS protocol). Representative plots are gated on γδ T cells.

Techniques Used: Enzyme-linked Immunospot, Quantitation Assay, Mouse Assay, Cell Culture, Staining

8) Product Images from "Elevated expression of interleukin-21 and its correlation to T-cell subpopulation in patients with ulcerative colitis"

Article Title: Elevated expression of interleukin-21 and its correlation to T-cell subpopulation in patients with ulcerative colitis

Journal: Central-European Journal of Immunology

doi: 10.5114/ceji.2015.54595

The percentages of circulating CD3 + CD8 – IL21 + T cells, Th17 cells, Th1 cells and Tc1 cells in representative UC patients and controls. Heparinized peripheral whole blood from all subjects was stimulated with PMA, ionomycin and monensin for 4 h, and then stained with labeled antibodies as described in methods. ( A ) and ( B ): Representative IL-21 expression in CD3 + CD8 – T subsets from UC patients and controls is shown. ( C ) and ( D ): Representative IL-17 expression in CD3 + CD8 – T subsets from UC patients and controls is shown. ( E ) and ( F ): Representative IFN-γ expression in CD3 + CD8 – T subsets (CD4 + T subsets) and CD + CD8 + T subsets from UC patients and controls is shown
Figure Legend Snippet: The percentages of circulating CD3 + CD8 – IL21 + T cells, Th17 cells, Th1 cells and Tc1 cells in representative UC patients and controls. Heparinized peripheral whole blood from all subjects was stimulated with PMA, ionomycin and monensin for 4 h, and then stained with labeled antibodies as described in methods. ( A ) and ( B ): Representative IL-21 expression in CD3 + CD8 – T subsets from UC patients and controls is shown. ( C ) and ( D ): Representative IL-17 expression in CD3 + CD8 – T subsets from UC patients and controls is shown. ( E ) and ( F ): Representative IFN-γ expression in CD3 + CD8 – T subsets (CD4 + T subsets) and CD + CD8 + T subsets from UC patients and controls is shown

Techniques Used: Staining, Labeling, Expressing

9) Product Images from "TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore"

Article Title: TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197622

The phosphorylation state of TRESK does not influence the kinetics of Ba 2+ block. Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K + -free bath solution to a high K + solution (solution changes are marked with bars above the graphs). Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. TRESK channels were either dephosphorylated by application of 0.5 μM ionomycin to the bath solution before recording or phosphorylated by application of purified MARK2 (16 μg/ml), 30 U/ml PKA (1 mM cAMP and 1 mM DTT was added to ensure the enzymatic activity of PKA) and 2 mM ATP before the initiation of the Ba 2+ block. A, Representative recording, TRESK channels were dephosphorylated before patch excision by application of 0.5 μM ionomycin to the bath solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, Representative recording, TRESK channels were phosphorylated by perfusing the intracellular side of the patch with a bath solution containing both kinases (purified MARK2, PKA, 1 mM cAMP, 1 mM DTT and 2 mM ATP) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). C, The kinetics of TRESK current inhibition by Ba 2+ were determined for both the phosphorylated and dephosphorylated channel (n = 6 and 5 patches). The average normalized curves for both groups are plotted. The inset shows the onset of Ba 2+ with a higher temporal resolution. D, The current recordings of the Ba 2+ block recorded for both the phosphorylated and dephosphorylated groups were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. The difference between the groups was not statistically significant (Student’s t test).
Figure Legend Snippet: The phosphorylation state of TRESK does not influence the kinetics of Ba 2+ block. Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K + -free bath solution to a high K + solution (solution changes are marked with bars above the graphs). Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. TRESK channels were either dephosphorylated by application of 0.5 μM ionomycin to the bath solution before recording or phosphorylated by application of purified MARK2 (16 μg/ml), 30 U/ml PKA (1 mM cAMP and 1 mM DTT was added to ensure the enzymatic activity of PKA) and 2 mM ATP before the initiation of the Ba 2+ block. A, Representative recording, TRESK channels were dephosphorylated before patch excision by application of 0.5 μM ionomycin to the bath solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, Representative recording, TRESK channels were phosphorylated by perfusing the intracellular side of the patch with a bath solution containing both kinases (purified MARK2, PKA, 1 mM cAMP, 1 mM DTT and 2 mM ATP) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). C, The kinetics of TRESK current inhibition by Ba 2+ were determined for both the phosphorylated and dephosphorylated channel (n = 6 and 5 patches). The average normalized curves for both groups are plotted. The inset shows the onset of Ba 2+ with a higher temporal resolution. D, The current recordings of the Ba 2+ block recorded for both the phosphorylated and dephosphorylated groups were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. The difference between the groups was not statistically significant (Student’s t test).

Techniques Used: Blocking Assay, Purification, Activity Assay, Inhibition

10) Product Images from "Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells"

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells

Journal: Nature Communications

doi: 10.1038/s41467-019-09431-3

Shp-2 is largely dispensable for NK cell effector functions. a Graphs depict percentages of CD94 + , Ly49A + , Ly49G2 + , Ly49I + , Ly49D + , Ly49H + , and KLRG1 + splenic NK cells (gated as NK1.1 + CD3/CD19 − ) from Ncr1 Ki Ptpn11 wt/wt (white), Ncr1 Ki Ptpn11 fl/fl (dark gray), and B2m −/− (green) mice. b Graph and a representative cytometric plot illustrate the production of granzyme A and B by splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) after phorbol 12-myristate 13-acetate and ionomycin stimulation (PMA/Iono). c NK cells isolated from polyinosinic:polycytidylic acid (polyI:C)-treated Ncr1 Ki Ptpn11 wt/wt or Ncr1 Ki Ptpn11 fl/fl mice were plated with RMA, RMA-S, or RMA-H60 cells at the indicated ratios. The graph depicts percentage killing of target cells, as measured by quantifying PI − living target cells. d Naive splenocytes from Ncr1 Tg Ptpn11 fl/fl (light gray) mice or heterozygote littermate controls (white) were incubated with RMA cells or RMA-m157 cells (expression of m157 is illustrated in the graph on the left). Percentage of YFP + Ly49H + CD107α + NK cells in each group was determined by flow cytometry (illustrated in the graph on the right). a , b Results represent the mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl or B2m −/− ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) mice per genotype and are representative of at least three ( a ) and two ( b ) independent experiments. c , d Results represent the mean ± SD of n = 3 ( c ), n = 4 (for non-stimulated conditions) and n = 5 (for RMA conditions) ( d ) technical replicates and are representative of at least two ( c ) and three ( d ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file
Figure Legend Snippet: Shp-2 is largely dispensable for NK cell effector functions. a Graphs depict percentages of CD94 + , Ly49A + , Ly49G2 + , Ly49I + , Ly49D + , Ly49H + , and KLRG1 + splenic NK cells (gated as NK1.1 + CD3/CD19 − ) from Ncr1 Ki Ptpn11 wt/wt (white), Ncr1 Ki Ptpn11 fl/fl (dark gray), and B2m −/− (green) mice. b Graph and a representative cytometric plot illustrate the production of granzyme A and B by splenic NK cells (gated as NK1.1 + NKp46 + CD3/CD19 − ) after phorbol 12-myristate 13-acetate and ionomycin stimulation (PMA/Iono). c NK cells isolated from polyinosinic:polycytidylic acid (polyI:C)-treated Ncr1 Ki Ptpn11 wt/wt or Ncr1 Ki Ptpn11 fl/fl mice were plated with RMA, RMA-S, or RMA-H60 cells at the indicated ratios. The graph depicts percentage killing of target cells, as measured by quantifying PI − living target cells. d Naive splenocytes from Ncr1 Tg Ptpn11 fl/fl (light gray) mice or heterozygote littermate controls (white) were incubated with RMA cells or RMA-m157 cells (expression of m157 is illustrated in the graph on the left). Percentage of YFP + Ly49H + CD107α + NK cells in each group was determined by flow cytometry (illustrated in the graph on the right). a , b Results represent the mean ± SEM of n = 4 ( Ncr1 Ki Ptpn11 fl/fl or B2m −/− ) and n = 6 ( Ncr1 Ki Ptpn11 wt/wt ) mice per genotype and are representative of at least three ( a ) and two ( b ) independent experiments. c , d Results represent the mean ± SD of n = 3 ( c ), n = 4 (for non-stimulated conditions) and n = 5 (for RMA conditions) ( d ) technical replicates and are representative of at least two ( c ) and three ( d ) independent experiments. Statistical comparisons are shown; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, NS, non-significant; Student’s t -test. Source data are provided as a Source Data file

Techniques Used: Mouse Assay, Isolation, Incubation, Expressing, Flow Cytometry, Cytometry

11) Product Images from "Epinephrine stimulation of anion secretion in the Calu-3 serous cell model"

Article Title: Epinephrine stimulation of anion secretion in the Calu-3 serous cell model

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.00190.2013

Effect of ionomycin pretreatment on the epinephrine-stimulated ion transport in the Calu-3 cell line. Confluent monolayers of Calu-3 cells were grown on permeable Transwell membranes for 14 days, excised, mounted in an Ussing chamber, and allowed to develop
Figure Legend Snippet: Effect of ionomycin pretreatment on the epinephrine-stimulated ion transport in the Calu-3 cell line. Confluent monolayers of Calu-3 cells were grown on permeable Transwell membranes for 14 days, excised, mounted in an Ussing chamber, and allowed to develop

Techniques Used:

12) Product Images from "The Profile of T Helper Subsets in Bone Marrow Microenvironment Is Distinct for Different Stages of Acute Myeloid Leukemia Patients and Chemotherapy Partly Ameliorates These Variations"

Article Title: The Profile of T Helper Subsets in Bone Marrow Microenvironment Is Distinct for Different Stages of Acute Myeloid Leukemia Patients and Chemotherapy Partly Ameliorates These Variations

Journal: PLoS ONE

doi: 10.1371/journal.pone.0131761

The percentage of Th22 cells in representative patients with ND, CR, relapsed-refractory AML patients or in controls. Heparinized bone marrow from all subjects was stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h or BM mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and then stained with labeled antibodies, as described in the “Materials and methods” section. A. Lymphocytes were gated by flow cytometry. B. The percentage of CD4 + IFN-γ - T cells in AML patients and controls. C, D, E, F. The percentage of Th22 (CD4 + IFN-γ - IL-17 - IL-22 + ) cells in AML patients and controls.
Figure Legend Snippet: The percentage of Th22 cells in representative patients with ND, CR, relapsed-refractory AML patients or in controls. Heparinized bone marrow from all subjects was stimulated with phorbol myristate acetate, ionomycin, and monensin for 4 h or BM mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and then stained with labeled antibodies, as described in the “Materials and methods” section. A. Lymphocytes were gated by flow cytometry. B. The percentage of CD4 + IFN-γ - T cells in AML patients and controls. C, D, E, F. The percentage of Th22 (CD4 + IFN-γ - IL-17 - IL-22 + ) cells in AML patients and controls.

Techniques Used: Isolation, Gradient Centrifugation, Staining, Labeling, Flow Cytometry, Cytometry

13) Product Images from "The LQLP Calcineurin Docking Site Is a Major Determinant of the Calcium-dependent Activation of Human TRESK Background K+ Channel *"

Article Title: The LQLP Calcineurin Docking Site Is a Major Determinant of the Calcium-dependent Activation of Human TRESK Background K+ Channel *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.577684

The AQAP mutation slows down the activation of human TRESK evoked by high concentration of ionomycin, but the inhibitory kinase reaction is not affected by this mutation. A , cells expressing wild type or AQAP mutant TRESK channels were stimulated with
Figure Legend Snippet: The AQAP mutation slows down the activation of human TRESK evoked by high concentration of ionomycin, but the inhibitory kinase reaction is not affected by this mutation. A , cells expressing wild type or AQAP mutant TRESK channels were stimulated with

Techniques Used: Mutagenesis, Activation Assay, Concentration Assay, Expressing

In mouse TRESK, the AQAP mutation also interferes with the calcium-dependent activation. A , average currents of two groups of oocytes expressing wild type or AQAP mutant mouse TRESK are plotted. The cells were stimulated with ionomycin ( Iono. , 0.5 μ
Figure Legend Snippet: In mouse TRESK, the AQAP mutation also interferes with the calcium-dependent activation. A , average currents of two groups of oocytes expressing wild type or AQAP mutant mouse TRESK are plotted. The cells were stimulated with ionomycin ( Iono. , 0.5 μ

Techniques Used: Mutagenesis, Activation Assay, Expressing

14) Product Images from "Importance of Th22 Cell Disequilibrium in Immune Thrombocytopenic Purpura"

Article Title: Importance of Th22 Cell Disequilibrium in Immune Thrombocytopenic Purpura

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.912528

Quantification of circulating CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells in each group. ( A–C ) Representative four-color dot plot analyses of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells after stimulation with PMA and ionomycin. ( D ) Frequency of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group. Results are represented as the mean ±SD (** p
Figure Legend Snippet: Quantification of circulating CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells in each group. ( A–C ) Representative four-color dot plot analyses of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells after stimulation with PMA and ionomycin. ( D ) Frequency of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group. Results are represented as the mean ±SD (** p

Techniques Used:

15) Product Images from "Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain"

Article Title: Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.127

Continuous GlyR α 3K 185L activation does not affect resting [Ca 2+ ] i . Intracellular Ca 2+ concentration at rest ([Ca 2+ ] i ) was determined according to the procedure described by Jung et al. 20 ( a ) Neurons were loaded 2–3 days after transfection with fura-2. Red and gray traces correspond to signals obtained from a GlyR α 3K 185L -positive neuron (red) and non-transfected neurons (gray) in the neighborhood of the transfected neuron in the same viewfield. A solution with 50 mM KCl was applied to monitor the viability of the cells according to their response with regard to fura-2 signals. Cells that did not respond to 50 mM KCl with changes in the F 340 /F 380 ratio were not included in the determination of resting [Ca 2+ ] i . Resting [Ca 2+ ] i was determined within the 10 s (marked with a yellow bar) prior to the application of 50 mM KCl. To obtain minimal (0 mM) and maximal (10 mM) Ca 2+ signals for calibration, cells were permeabilized with either 10 μ M ionomycin or 10 μ M 4-bromo-antibiotic A23187. 2 mM Mn 2+ were applied to quench the signal at the end of each experiment and to obtain background fluorescence that was subtracted from all F340 and F380 values. ( b ) GlyR α 3K 185L expression and activation in the presence of 10 μ M glycine does not affect resting [Ca 2+ ] i . Numbers in the bar graphs indicate the number of neurons analyzed
Figure Legend Snippet: Continuous GlyR α 3K 185L activation does not affect resting [Ca 2+ ] i . Intracellular Ca 2+ concentration at rest ([Ca 2+ ] i ) was determined according to the procedure described by Jung et al. 20 ( a ) Neurons were loaded 2–3 days after transfection with fura-2. Red and gray traces correspond to signals obtained from a GlyR α 3K 185L -positive neuron (red) and non-transfected neurons (gray) in the neighborhood of the transfected neuron in the same viewfield. A solution with 50 mM KCl was applied to monitor the viability of the cells according to their response with regard to fura-2 signals. Cells that did not respond to 50 mM KCl with changes in the F 340 /F 380 ratio were not included in the determination of resting [Ca 2+ ] i . Resting [Ca 2+ ] i was determined within the 10 s (marked with a yellow bar) prior to the application of 50 mM KCl. To obtain minimal (0 mM) and maximal (10 mM) Ca 2+ signals for calibration, cells were permeabilized with either 10 μ M ionomycin or 10 μ M 4-bromo-antibiotic A23187. 2 mM Mn 2+ were applied to quench the signal at the end of each experiment and to obtain background fluorescence that was subtracted from all F340 and F380 values. ( b ) GlyR α 3K 185L expression and activation in the presence of 10 μ M glycine does not affect resting [Ca 2+ ] i . Numbers in the bar graphs indicate the number of neurons analyzed

Techniques Used: Activation Assay, Concentration Assay, Transfection, Fluorescence, Expressing

16) Product Images from "Decreased Gaq expression in T cells correlates with enhanced cytokine production and disease activity in systemic lupus erythematosus"

Article Title: Decreased Gaq expression in T cells correlates with enhanced cytokine production and disease activity in systemic lupus erythematosus

Journal: Oncotarget

doi: 10.18632/oncotarget.13903

Gαq deletion promotes the differentiation of T-helper cells The expression of IFN-γ A., IL-4 B., and IL-17 C. was studied by flow cytometry in Gαq knockout (Gnaq-/-) and wild type (WT) mice-derived splenic CD4+ T cells stimulated with PMA, ionomycin, and BFA. Data from three independent experiments are presented as mean ± SD.
Figure Legend Snippet: Gαq deletion promotes the differentiation of T-helper cells The expression of IFN-γ A., IL-4 B., and IL-17 C. was studied by flow cytometry in Gαq knockout (Gnaq-/-) and wild type (WT) mice-derived splenic CD4+ T cells stimulated with PMA, ionomycin, and BFA. Data from three independent experiments are presented as mean ± SD.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Knock-Out, Mouse Assay, Derivative Assay

17) Product Images from "Liver receptor homolog-1 (NR5a2) regulates CD95/Fas ligand transcription and associated T-cell effector functions"

Article Title: Liver receptor homolog-1 (NR5a2) regulates CD95/Fas ligand transcription and associated T-cell effector functions

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.173

LRH-1 inhibition by 3d2 restricts FASLG promoter activity and expression. ( a ) Human FasL mRNA expression of Jurkat lT cells after treatment with PMA/ionomycin (50/500 ng/ml) for 18 h was determined using quantitative PCR. Mean values of triplicates±S.D. are shown. ( b ) Jurkat lT cells were co-transfected with a FASL luciferase promoter reporter, and control plasmids (pcDNA) or an LRH-1 expression plasmid. Cells were then stimulated with PMA/ionomycin (50/500 ng/ml) and the indicated concentrations of 3d2 for 18 h. Luciferase reporter activity was measured and normalized to luciferase control plasmid. Mean values of triplicates±S.D. of a representative experiment ( n =3) are shown (unpaired t -test; * P
Figure Legend Snippet: LRH-1 inhibition by 3d2 restricts FASLG promoter activity and expression. ( a ) Human FasL mRNA expression of Jurkat lT cells after treatment with PMA/ionomycin (50/500 ng/ml) for 18 h was determined using quantitative PCR. Mean values of triplicates±S.D. are shown. ( b ) Jurkat lT cells were co-transfected with a FASL luciferase promoter reporter, and control plasmids (pcDNA) or an LRH-1 expression plasmid. Cells were then stimulated with PMA/ionomycin (50/500 ng/ml) and the indicated concentrations of 3d2 for 18 h. Luciferase reporter activity was measured and normalized to luciferase control plasmid. Mean values of triplicates±S.D. of a representative experiment ( n =3) are shown (unpaired t -test; * P

Techniques Used: Inhibition, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation

18) Product Images from "Effector T helper cell populations are elevated in the bone marrow of rheumatoid arthritis patients and correlate with disease severity"

Article Title: Effector T helper cell populations are elevated in the bone marrow of rheumatoid arthritis patients and correlate with disease severity

Journal: Scientific Reports

doi: 10.1038/s41598-017-05014-8

Frequency of Th22 based on cytokine patterns after in vitro activation by PMA/ionomycin in short-term cultures. ( A ) Flow cytometry for the detection of the frequency of Th22. ( B ) The percentages of Th22 in peripheral blood from rheumatoid arthritis patients (RAPB), osteoarthritis patients(OAPB) () and healthy controls (HCPB). ( C ) The percentages of Th22 in bone marrow blood from rheumatoid arthritis patients (RABM), osteoarthritis patients(OABM) and healthy controls (HCBM). ( D ) The percentages of Th22 in peripheral blood (RAPB) and bone marrow blood (RABM) from rheumatoid arthritis patients. *P
Figure Legend Snippet: Frequency of Th22 based on cytokine patterns after in vitro activation by PMA/ionomycin in short-term cultures. ( A ) Flow cytometry for the detection of the frequency of Th22. ( B ) The percentages of Th22 in peripheral blood from rheumatoid arthritis patients (RAPB), osteoarthritis patients(OAPB) () and healthy controls (HCPB). ( C ) The percentages of Th22 in bone marrow blood from rheumatoid arthritis patients (RABM), osteoarthritis patients(OABM) and healthy controls (HCBM). ( D ) The percentages of Th22 in peripheral blood (RAPB) and bone marrow blood (RABM) from rheumatoid arthritis patients. *P

Techniques Used: In Vitro, Activation Assay, Flow Cytometry, Cytometry

19) Product Images from "Up-Regulation of GITRL on Dendritic Cells by WGP Improves Anti-Tumor Immunity in Murine Lewis Lung Carcinoma"

Article Title: Up-Regulation of GITRL on Dendritic Cells by WGP Improves Anti-Tumor Immunity in Murine Lewis Lung Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0046936

WGP induces enhanced CTL priming in vivo . Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in Materials and Methods . (A, B) Single cell suspensions prepared from spleens (A) and draining lymph nodes (B) were stimulated with PMA plus ionomycin and stained intracellular IFN-γ. Cells were gated on CD3 + CD8 + T cells. Culture supernatants from splenocytes and lymphoid cells were collected and assayed for IFN-γ using ELISA. (C) Tumor specimens from each group were prepared for single cell suspensions. Cells were stained with mAbs against CD3, CD8 and were assessed by flow cytometry. Cells were gated on CD3 + T cells. RNAs from tumor specimens were extracted and qRT-PCR was performed for IFN-γ. Results are expressed as mean ± SD. **P
Figure Legend Snippet: WGP induces enhanced CTL priming in vivo . Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in Materials and Methods . (A, B) Single cell suspensions prepared from spleens (A) and draining lymph nodes (B) were stimulated with PMA plus ionomycin and stained intracellular IFN-γ. Cells were gated on CD3 + CD8 + T cells. Culture supernatants from splenocytes and lymphoid cells were collected and assayed for IFN-γ using ELISA. (C) Tumor specimens from each group were prepared for single cell suspensions. Cells were stained with mAbs against CD3, CD8 and were assessed by flow cytometry. Cells were gated on CD3 + T cells. RNAs from tumor specimens were extracted and qRT-PCR was performed for IFN-γ. Results are expressed as mean ± SD. **P

Techniques Used: CTL Assay, In Vivo, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR

20) Product Images from "The B? and B? Regulatory Subunits of PP2A are Necessary for Assembly of the CaMKIVoPP2A Signaling Complex"

Article Title: The B? and B? Regulatory Subunits of PP2A are Necessary for Assembly of the CaMKIVoPP2A Signaling Complex

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.06.062

Inhibition of PP2A enhances ionomycin-induced phosphorylation of endogenous but not ectopic CaMKIV phosphorylation
Figure Legend Snippet: Inhibition of PP2A enhances ionomycin-induced phosphorylation of endogenous but not ectopic CaMKIV phosphorylation

Techniques Used: Inhibition

21) Product Images from "The parathyroid hormone family member TIP39 interacts with sarco/endoplasmic reticulum Ca2+- ATPase activity by influencing calcium homeostasis"

Article Title: The parathyroid hormone family member TIP39 interacts with sarco/endoplasmic reticulum Ca2+- ATPase activity by influencing calcium homeostasis

Journal: Experimental dermatology

doi: 10.1111/exd.13294

TIP39 increased intracellular calcium from ER and enhanced SERCA activity after extracellular Ca 2+ supplementation (a) Intracellular calcium change by 10μM TIP39 was monitored in the absence of extracellular calcium or (b) low calcium (0.05mM CaCl 2 ) contained-balanced salt solution. Both (a) and (b) samples were incubated for 70min in those salt solution before monitoring. For complete removal of extracellular calcium, 150 μM EGTA was added in the calcium-free solution of (a). (c) The bar graph shows comparison of % [Ca 2+ ]i increase of (a) and (b). The data were randomly extracted at 5 time points in 10 min after TIP39 stimulation. (Each group n=5, total n=25) (d) An experimental schema of (e), which analyzes ER Ca 2+ content after TIP39 pretreatment. (e) ER Ca 2+ content was monitored after 5 μM ionomycin stimulation with or without 30min pretreatment of 10μM TIP39 in the absence of extracellular calcium. (f) An experimental schema of (g), which analyzes ER Ca 2+ content after extracellular calcium rescue. (g) ER Ca 2+ content was monitored after stimulation of 5 μM ionomycin with 10min rescue of 2.5 mM CaCl 2 . Fold change (%) = (F-F0) / F0×100, F0 was the baseline calculated by averaging five time points just prior to the application of the stimulus. Data are means ± SEM, n=5 and are representative data from two independent experiments.*p
Figure Legend Snippet: TIP39 increased intracellular calcium from ER and enhanced SERCA activity after extracellular Ca 2+ supplementation (a) Intracellular calcium change by 10μM TIP39 was monitored in the absence of extracellular calcium or (b) low calcium (0.05mM CaCl 2 ) contained-balanced salt solution. Both (a) and (b) samples were incubated for 70min in those salt solution before monitoring. For complete removal of extracellular calcium, 150 μM EGTA was added in the calcium-free solution of (a). (c) The bar graph shows comparison of % [Ca 2+ ]i increase of (a) and (b). The data were randomly extracted at 5 time points in 10 min after TIP39 stimulation. (Each group n=5, total n=25) (d) An experimental schema of (e), which analyzes ER Ca 2+ content after TIP39 pretreatment. (e) ER Ca 2+ content was monitored after 5 μM ionomycin stimulation with or without 30min pretreatment of 10μM TIP39 in the absence of extracellular calcium. (f) An experimental schema of (g), which analyzes ER Ca 2+ content after extracellular calcium rescue. (g) ER Ca 2+ content was monitored after stimulation of 5 μM ionomycin with 10min rescue of 2.5 mM CaCl 2 . Fold change (%) = (F-F0) / F0×100, F0 was the baseline calculated by averaging five time points just prior to the application of the stimulus. Data are means ± SEM, n=5 and are representative data from two independent experiments.*p

Techniques Used: Activity Assay, Incubation

TIP39 increased cytosolic calcium in ATP2A2 silenced-keratinocyte, but the reaction was incomplete compared with control (a) NHEK were transfected siRNA for 72h then analyzed by qRT-PCR and (b) western blotting of ATP2A2 and GAPDH. GAPDH is used for housekeeping gene and protein. (c) Immunofluorescence staining of desmoplakin in siRNA transfected NHEK. Scale bars, 20μm. (d) Basic fluo-4 signal of siRNA transfected-NHEK in the absence of extracellular calcium. (e) ER calcium content was monitored after 5μM ionomycin stimulation in the absence of extracellular calcium. (f) Cytoplasmic calcium was monitored after stimulation of 25μM TIP39 in the absence of extracellular calcium. (g) Intracellular calcium imaging of siRNA transfected-keratinocytes. Cells were treated by 10μM TIP39, 5μM thapsigargin or 2mM CaCl 2 and 10μM ionomycin, then checked by microscopy at 5min and (h) 30min after stimulation. Scale bars, 20μm. Fold change (%) = (F-F0) / F0×100, Data are means ± SEM of biological replicates, n=3 (qRT-PCR) or n=5 (calcium assay) and are representative data from at least two independent experiments. .*p
Figure Legend Snippet: TIP39 increased cytosolic calcium in ATP2A2 silenced-keratinocyte, but the reaction was incomplete compared with control (a) NHEK were transfected siRNA for 72h then analyzed by qRT-PCR and (b) western blotting of ATP2A2 and GAPDH. GAPDH is used for housekeeping gene and protein. (c) Immunofluorescence staining of desmoplakin in siRNA transfected NHEK. Scale bars, 20μm. (d) Basic fluo-4 signal of siRNA transfected-NHEK in the absence of extracellular calcium. (e) ER calcium content was monitored after 5μM ionomycin stimulation in the absence of extracellular calcium. (f) Cytoplasmic calcium was monitored after stimulation of 25μM TIP39 in the absence of extracellular calcium. (g) Intracellular calcium imaging of siRNA transfected-keratinocytes. Cells were treated by 10μM TIP39, 5μM thapsigargin or 2mM CaCl 2 and 10μM ionomycin, then checked by microscopy at 5min and (h) 30min after stimulation. Scale bars, 20μm. Fold change (%) = (F-F0) / F0×100, Data are means ± SEM of biological replicates, n=3 (qRT-PCR) or n=5 (calcium assay) and are representative data from at least two independent experiments. .*p

Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Imaging, Microscopy, Calcium Assay

22) Product Images from "Niclosamide repurposed for the treatment of inflammatory airway disease"

Article Title: Niclosamide repurposed for the treatment of inflammatory airway disease

Journal: JCI Insight

doi: 10.1172/jci.insight.128414

Inhibition of TMEM16A and TMEM16F by niclosamide. ( A ) TMEM16A whole-cell currents in TMEM16A-overexpressing HEK293 cells. The purinergic agonist UTP (100 μM) was used to activate TMEM16A. UTP-induced currents were inhibited by niclosamide (1 μM). ( B and C ) Concentration-dependent inhibition of TMEM16A by niclosamide ( n = 5–7 cells). ( D ) Current/voltage relationship showing inhibition of TMEM16F expressed in HEK293 cells by niclosamide ( n = 5 cells each). ( E ) Inhibition of endogenous TMEM16A/F expressed in HT-29 cells examined by iodide quenching. Rate of YFP quenching (AU/s) when applying 20 mM iodide to the extracellular bath solution. HT-29 cells stably overexpressing YFP were stimulated with 1 μM ionomycin. Per well, 100,000 cells were seeded ( n = 6–8 wells for each concentration). Inset: Western blot indicating expression of TMEM16F in HT-29 cells. Data are reported as mean ± SEM. *Significant activation ( P
Figure Legend Snippet: Inhibition of TMEM16A and TMEM16F by niclosamide. ( A ) TMEM16A whole-cell currents in TMEM16A-overexpressing HEK293 cells. The purinergic agonist UTP (100 μM) was used to activate TMEM16A. UTP-induced currents were inhibited by niclosamide (1 μM). ( B and C ) Concentration-dependent inhibition of TMEM16A by niclosamide ( n = 5–7 cells). ( D ) Current/voltage relationship showing inhibition of TMEM16F expressed in HEK293 cells by niclosamide ( n = 5 cells each). ( E ) Inhibition of endogenous TMEM16A/F expressed in HT-29 cells examined by iodide quenching. Rate of YFP quenching (AU/s) when applying 20 mM iodide to the extracellular bath solution. HT-29 cells stably overexpressing YFP were stimulated with 1 μM ionomycin. Per well, 100,000 cells were seeded ( n = 6–8 wells for each concentration). Inset: Western blot indicating expression of TMEM16F in HT-29 cells. Data are reported as mean ± SEM. *Significant activation ( P

Techniques Used: Inhibition, Concentration Assay, Stable Transfection, Western Blot, Expressing, Activation Assay

The TMEM16 inhibitor niflumic acid attenuates inflammatory airway disease. ( A and B ) OVA sensitization induced goblet cell metaplasia as indicated by Alcian blue positivity. Exposure to carbachol (CCH, 25 mg/mL, nebulizer) induced release of mucus and airway contraction ( n = 3 mice/8–20 airway sections). Scale bars: 20 μm. Preexposure to the TMEM16A inhibitor niflumic acid (NFA; 0.5 mg/kg/d, intratracheal application for 3 days) strongly attenuated mucus production and CCH-induced airway contraction ( A–C ). ( C ) Cross section of airways indicating airway relaxation by NFA (3 mice/8–13 airway sections). ( D–F ) Whole-cell currents obtained in patch-clamp experiments with HEK293 cells expressing TMEM16A or TMEM16F. Currents were activated by 1 μM ionomycin and inhibited by NFA (20 μM) ( n = 5–6 cells). Scale bars: 20 μm. Data are reported as mean ± SEM. # Significant increase compared with control ( P
Figure Legend Snippet: The TMEM16 inhibitor niflumic acid attenuates inflammatory airway disease. ( A and B ) OVA sensitization induced goblet cell metaplasia as indicated by Alcian blue positivity. Exposure to carbachol (CCH, 25 mg/mL, nebulizer) induced release of mucus and airway contraction ( n = 3 mice/8–20 airway sections). Scale bars: 20 μm. Preexposure to the TMEM16A inhibitor niflumic acid (NFA; 0.5 mg/kg/d, intratracheal application for 3 days) strongly attenuated mucus production and CCH-induced airway contraction ( A–C ). ( C ) Cross section of airways indicating airway relaxation by NFA (3 mice/8–13 airway sections). ( D–F ) Whole-cell currents obtained in patch-clamp experiments with HEK293 cells expressing TMEM16A or TMEM16F. Currents were activated by 1 μM ionomycin and inhibited by NFA (20 μM) ( n = 5–6 cells). Scale bars: 20 μm. Data are reported as mean ± SEM. # Significant increase compared with control ( P

Techniques Used: Mouse Assay, Patch Clamp, Expressing

23) Product Images from "Aberrant T Helper 17 Cells and Related Cytokines in Bone Marrow Microenvironment of Patients with Acute Myeloid Leukemia"

Article Title: Aberrant T Helper 17 Cells and Related Cytokines in Bone Marrow Microenvironment of Patients with Acute Myeloid Leukemia

Journal: Clinical and Developmental Immunology

doi: 10.1155/2013/915873

Th subsets and their related cytokines in ND, relapsed-refractory, and CR AML patients and controls. (a) The percentage of BM Th17 cells was significantly decreased in ND AML patients compared with CR patients or controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. (b) The level of BM plasma IL-17A showed the decreased trend in the ND, relapsed-refractory, or CR AML patients compared with controls, though no statistical significance exists. (c) The percentage of BM Th1 cells was significantly decreased in ND AML patients compared with relapsed-refractory or CR patients or controls. (d) The level of BM plasma IFN- γ was decreased in the ND AML patients compared with CR patients.
Figure Legend Snippet: Th subsets and their related cytokines in ND, relapsed-refractory, and CR AML patients and controls. (a) The percentage of BM Th17 cells was significantly decreased in ND AML patients compared with CR patients or controls after stimulation with phorbol myristate acetate, ionomycin, and monensin for 4 h. (b) The level of BM plasma IL-17A showed the decreased trend in the ND, relapsed-refractory, or CR AML patients compared with controls, though no statistical significance exists. (c) The percentage of BM Th1 cells was significantly decreased in ND AML patients compared with relapsed-refractory or CR patients or controls. (d) The level of BM plasma IFN- γ was decreased in the ND AML patients compared with CR patients.

Techniques Used:

24) Product Images from "Elevated Frequencies of Circulating Th22 Cell in Addition to Th17 Cell and Th17/Th1 Cell in Patients with Acute Coronary Syndrome"

Article Title: Elevated Frequencies of Circulating Th22 Cell in Addition to Th17 Cell and Th17/Th1 Cell in Patients with Acute Coronary Syndrome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071466

Flow cytometric analysis of Th1 cells, Th22 cells and CD4 + IFNγ − IL17 + IL-22 + cells. Peripheral blood from patients with AMI, UA, SA and HC subjects were stimulated with PMA, ionomycin and monensin for 4 h, and then stained with labeled antibodies as described in Methods . A, Gating strategies and representative flow cytometry dot plot results of each group. Lymphocytes were gated in R1 by forward and side scatter gating. These cells were analyzed for IFN-γ producing and CD4 expression T cells. CD4 + IFN-γ − cells were gated in R2 and analyzed for IL-17 and IL-22 producing T cells. Numbers represent the percentage of cells in the quadrants. B, Comparison of the percentages of circulating Th22 cells (left panel, % of CD4 + IFN-γ − cells), Th1 cells (middle panel, % of total lymphocytes) and CD4 + IFNγ − IL17 + IL-22 + cells (right panel, % of CD4 + IFN-γ − cells) from AMI, UA, SA patients and healthy controls. (* = P
Figure Legend Snippet: Flow cytometric analysis of Th1 cells, Th22 cells and CD4 + IFNγ − IL17 + IL-22 + cells. Peripheral blood from patients with AMI, UA, SA and HC subjects were stimulated with PMA, ionomycin and monensin for 4 h, and then stained with labeled antibodies as described in Methods . A, Gating strategies and representative flow cytometry dot plot results of each group. Lymphocytes were gated in R1 by forward and side scatter gating. These cells were analyzed for IFN-γ producing and CD4 expression T cells. CD4 + IFN-γ − cells were gated in R2 and analyzed for IL-17 and IL-22 producing T cells. Numbers represent the percentage of cells in the quadrants. B, Comparison of the percentages of circulating Th22 cells (left panel, % of CD4 + IFN-γ − cells), Th1 cells (middle panel, % of total lymphocytes) and CD4 + IFNγ − IL17 + IL-22 + cells (right panel, % of CD4 + IFN-γ − cells) from AMI, UA, SA patients and healthy controls. (* = P

Techniques Used: Flow Cytometry, Staining, Labeling, Cytometry, Expressing

Flow cytometric analysis of Th17 cells and Th17/ Th1 cells. Peripheral blood from patients with AMI, UA, SA and HC subjects were stimulated with PMA, ionomycin and monensin for 4 + cells were gated in R2 and analyzed for IL-17 and IFN-γ producing T cells. Numbers represent the percentage of cells in the quadrants. B, Comparison of the percentages of Th17 cells and Th17/Th1 cells in CD4 + cells from AMI, UA, SA patients and healthy controls. (* = P
Figure Legend Snippet: Flow cytometric analysis of Th17 cells and Th17/ Th1 cells. Peripheral blood from patients with AMI, UA, SA and HC subjects were stimulated with PMA, ionomycin and monensin for 4 + cells were gated in R2 and analyzed for IL-17 and IFN-γ producing T cells. Numbers represent the percentage of cells in the quadrants. B, Comparison of the percentages of Th17 cells and Th17/Th1 cells in CD4 + cells from AMI, UA, SA patients and healthy controls. (* = P

Techniques Used: Flow Cytometry

25) Product Images from "The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling"

Article Title: The paracaspase MALT1 cleaves HOIL1 reducing linear ubiquitination by LUBAC to dampen lymphocyte NF-κB signalling

Journal: Nature Communications

doi: 10.1038/ncomms9777

Validation of HOIL1 as a MALT1 substrate in vitro and in cells. ( a ) Concentration-dependent in vitro cleavage of recombinant human HOIL1 by recombinant human MALT1. Cleavage products were identified using anti-C-terminal FLAG and anti-HOIL1 N-terminal antibodies, N =2. ( b ) Immunoblot for HOIL1 cleavage after co-transfection of HOIL1 with CBM proteins: oncogenic mutant CARD11-(Leu244Pro)(L/P), BCL10 and MALT1-(FLAG-His 6 ), or catalytically inactive mutant MALT1-(Cys464Ala)(C/A) in HEK293FT cells (left panel), and effect of substitution by charge conserving cleavage site mutation HOIL1-(R165K) (right panel). ( c ) αV5 immunoblot for HOIL1-V5 and cleavage site mutated HOIL1-(Arg165Lys)(R/K)-V5 cleavage by lymphoma fusion protein cIAP2-MALT1-(WT) or the catalytically inactive cIAP2-MALT1-(Cys464Ala)(C/A) in HEK293FT cells. ( d ) Cell–cell contact aligned merged confocal microscopy image slices through the middle of B cells from a normal donor (+/+ N) and the patient ( mut / mut ) with and without pretreatment with soluble α-IgG prior to immobilization on α-IgG/IgM coated coverslips and staining. Monoclonal antibody-labelled HOIL1 (green) and MALT1 (red) are shown, yellow arrows indicate co-localization. Blue channel, 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar, 10 μm. Individual laser channels for three fields are in Supplementary Fig. 6 . ( e ) α-HOIL1 immunoblot for cleavage of endogenous HOIL1 in immortalized B cells from a normal donor (+/+ N) and brother (+/ mut B) after PMA/ionomycin stimulation with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =34. ( f ) α−HOIL1 immunoblot for HOIL1 cleavage in PMA/ionomycin-stimulated primary peripheral blood mononuclear cells (PBMCs) from the mother (+/ mut M), a normal donor (+/+N) and the patient ( mut / mut ) with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =9. ( g ) Patient ( MALT1 mut/mut ) skin haematoxylin and eosin staining of lymphocytic (LC) infiltrates (white arrows) in the upper dermis (D) surrounding vessels, and the basal layer of the overlying epidermis (E). Immunohistochemistry identified the lymphocytes as CD3 + T cells. ( h ) α-HOIL1 immunoblot of HOIL1 cleavage in PMA/ionomycin-stimulated primary CD4 + and CD8 + T cells from the mother (+/ mut M), a normal donor (+/+ N) and the patient ( mut / mut ), N =6. * Represents consistently observed nonspecific band. Tubulin, β-actin: loading controls.
Figure Legend Snippet: Validation of HOIL1 as a MALT1 substrate in vitro and in cells. ( a ) Concentration-dependent in vitro cleavage of recombinant human HOIL1 by recombinant human MALT1. Cleavage products were identified using anti-C-terminal FLAG and anti-HOIL1 N-terminal antibodies, N =2. ( b ) Immunoblot for HOIL1 cleavage after co-transfection of HOIL1 with CBM proteins: oncogenic mutant CARD11-(Leu244Pro)(L/P), BCL10 and MALT1-(FLAG-His 6 ), or catalytically inactive mutant MALT1-(Cys464Ala)(C/A) in HEK293FT cells (left panel), and effect of substitution by charge conserving cleavage site mutation HOIL1-(R165K) (right panel). ( c ) αV5 immunoblot for HOIL1-V5 and cleavage site mutated HOIL1-(Arg165Lys)(R/K)-V5 cleavage by lymphoma fusion protein cIAP2-MALT1-(WT) or the catalytically inactive cIAP2-MALT1-(Cys464Ala)(C/A) in HEK293FT cells. ( d ) Cell–cell contact aligned merged confocal microscopy image slices through the middle of B cells from a normal donor (+/+ N) and the patient ( mut / mut ) with and without pretreatment with soluble α-IgG prior to immobilization on α-IgG/IgM coated coverslips and staining. Monoclonal antibody-labelled HOIL1 (green) and MALT1 (red) are shown, yellow arrows indicate co-localization. Blue channel, 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar, 10 μm. Individual laser channels for three fields are in Supplementary Fig. 6 . ( e ) α-HOIL1 immunoblot for cleavage of endogenous HOIL1 in immortalized B cells from a normal donor (+/+ N) and brother (+/ mut B) after PMA/ionomycin stimulation with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =34. ( f ) α−HOIL1 immunoblot for HOIL1 cleavage in PMA/ionomycin-stimulated primary peripheral blood mononuclear cells (PBMCs) from the mother (+/ mut M), a normal donor (+/+N) and the patient ( mut / mut ) with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =9. ( g ) Patient ( MALT1 mut/mut ) skin haematoxylin and eosin staining of lymphocytic (LC) infiltrates (white arrows) in the upper dermis (D) surrounding vessels, and the basal layer of the overlying epidermis (E). Immunohistochemistry identified the lymphocytes as CD3 + T cells. ( h ) α-HOIL1 immunoblot of HOIL1 cleavage in PMA/ionomycin-stimulated primary CD4 + and CD8 + T cells from the mother (+/ mut M), a normal donor (+/+ N) and the patient ( mut / mut ), N =6. * Represents consistently observed nonspecific band. Tubulin, β-actin: loading controls.

Techniques Used: In Vitro, Concentration Assay, Recombinant, Cotransfection, Mutagenesis, Confocal Microscopy, Staining, Immunohistochemistry

N-terminal TAILS proteomics investigation of patient and control B cells reveal HOIL1 as a MALT1 substrate. ( a ) 10-plex TMT labelling scheme for TAILS and preTAILS shotgun proteomics analyses of immortalized B-cell lysates from the MALT1 mutant patient ( mut / mut ) and heterozygous brother (+/ mut B) and mother (+/ mut M) controls after 2 and 4 h stimulation with PMA/ionomycin (PMA/Iono) or solvent (control; N=2 ). Peptides were identified by MS/MS (FDR≤1.0%) and quantified by SPS-MS/MS/MS. ( b ) All unique peptides (including cyclization of glutamine and carry-over of unblocked peptides), unique proteins and unique N-terminal peptides ( N -acetylated and N-TMT labelled) identified by Byonic in the TAILS and shotgun proteomics analyses at an FDR of 1% at the protein level. Individual peptides were subsequently filtered at a probability > 0.99. ( c ) MS/MS spectrum of 166 GPLEPGPPKPGVPQEPGR 182 , the neo-N-terminal peptide of the carboxy-cleavage product of HOIL1 identified by TAILS. * Represents TMT-labelled residues. ( d ) Absolute intensity values of the 10-plex isobaric reporters from the spectrum in c of control (black) and 2 h PMA/ionomycin-stimulated (red) samples. ( e ) MALT1-cleavage site in HOIL1 identified by TAILS from the high reporter ratio of the neo-N-terminal peptide (red) comparing +/ mut to mut / mut samples both before (black bars) and after PMA/ionomycin stimulation (red bars; n =10). The natural protein N-terminal peptide of HOIL1 was identified by TAILS (red; n =3), whereas the nonprime side peptide of the cleavage site was identified by shotgun proteomics (blue; n =4). The figure is representative of independent experiments yielding similar results. * Represents TMT-labelled amino acid; Ac, N-terminal acetylation. ( f ) Sequence alignment of the MALT1-cleavage site in HOIL1 with all known MALT1 substrates; m, murine; h, human. Amino acids in all sequences that are identical (yellow), similar (green) and identical in some (blue) are shown.
Figure Legend Snippet: N-terminal TAILS proteomics investigation of patient and control B cells reveal HOIL1 as a MALT1 substrate. ( a ) 10-plex TMT labelling scheme for TAILS and preTAILS shotgun proteomics analyses of immortalized B-cell lysates from the MALT1 mutant patient ( mut / mut ) and heterozygous brother (+/ mut B) and mother (+/ mut M) controls after 2 and 4 h stimulation with PMA/ionomycin (PMA/Iono) or solvent (control; N=2 ). Peptides were identified by MS/MS (FDR≤1.0%) and quantified by SPS-MS/MS/MS. ( b ) All unique peptides (including cyclization of glutamine and carry-over of unblocked peptides), unique proteins and unique N-terminal peptides ( N -acetylated and N-TMT labelled) identified by Byonic in the TAILS and shotgun proteomics analyses at an FDR of 1% at the protein level. Individual peptides were subsequently filtered at a probability > 0.99. ( c ) MS/MS spectrum of 166 GPLEPGPPKPGVPQEPGR 182 , the neo-N-terminal peptide of the carboxy-cleavage product of HOIL1 identified by TAILS. * Represents TMT-labelled residues. ( d ) Absolute intensity values of the 10-plex isobaric reporters from the spectrum in c of control (black) and 2 h PMA/ionomycin-stimulated (red) samples. ( e ) MALT1-cleavage site in HOIL1 identified by TAILS from the high reporter ratio of the neo-N-terminal peptide (red) comparing +/ mut to mut / mut samples both before (black bars) and after PMA/ionomycin stimulation (red bars; n =10). The natural protein N-terminal peptide of HOIL1 was identified by TAILS (red; n =3), whereas the nonprime side peptide of the cleavage site was identified by shotgun proteomics (blue; n =4). The figure is representative of independent experiments yielding similar results. * Represents TMT-labelled amino acid; Ac, N-terminal acetylation. ( f ) Sequence alignment of the MALT1-cleavage site in HOIL1 with all known MALT1 substrates; m, murine; h, human. Amino acids in all sequences that are identical (yellow), similar (green) and identical in some (blue) are shown.

Techniques Used: Mutagenesis, Mass Spectrometry, Sequencing

MALT1 remodels LUBAC, releasing C-HOIL1 and reducing HOIP protein level. ( a ) Diagram of LUBAC subunits in relation to the MALT1-cleavage site in HOIL1 that generates N-HOIL1 and C-HOIL1. ( b ) Immunoblots for HOIP and SHARPIN showed MALT1 does not cleave these proteins when active CBM is coexpressed in HEK293 cells. ( c , d ) Assembly of LUBAC in HEK293FT cells by co-transfection of HOIL1-FLAG, SHARPIN-V5, HOIP-V5 and five times (5 × ) HOIP-V5 vector. Anti-FLAG (for HOIL1, N-HOIL1 and C-HOIL1) or anti-V5 (for HOIP) immunoprecipitation (IP) and immunoblotting (IB) for HOIL1, N-HOIL1 and C-HOIL1 us ing anti-FLAG or HOIP using anti-V5 in the immunoprecipitates showed HOIP interaction with HOIL1 and N-HOIL1 but not with C-HOIL1. ( e ) α-V5 IP of lysates of proteasome inhibitor (MG132) treated HEK293FT cells transfected with HOIP-V5, SHARPIN-V5 with or without co-transfection of HOIL1, and subsequent α-HOIP immunoblotting. N =3. ( f ) HOIP protein level in B cells from the brother (+/ mut B) and patient ( mut / mut ) on PMA/Ionomycin stimulation over time. β-Actin, loading control, N =2. Orange arrowheads indicate decreased HOIP levels.
Figure Legend Snippet: MALT1 remodels LUBAC, releasing C-HOIL1 and reducing HOIP protein level. ( a ) Diagram of LUBAC subunits in relation to the MALT1-cleavage site in HOIL1 that generates N-HOIL1 and C-HOIL1. ( b ) Immunoblots for HOIP and SHARPIN showed MALT1 does not cleave these proteins when active CBM is coexpressed in HEK293 cells. ( c , d ) Assembly of LUBAC in HEK293FT cells by co-transfection of HOIL1-FLAG, SHARPIN-V5, HOIP-V5 and five times (5 × ) HOIP-V5 vector. Anti-FLAG (for HOIL1, N-HOIL1 and C-HOIL1) or anti-V5 (for HOIP) immunoprecipitation (IP) and immunoblotting (IB) for HOIL1, N-HOIL1 and C-HOIL1 us ing anti-FLAG or HOIP using anti-V5 in the immunoprecipitates showed HOIP interaction with HOIL1 and N-HOIL1 but not with C-HOIL1. ( e ) α-V5 IP of lysates of proteasome inhibitor (MG132) treated HEK293FT cells transfected with HOIP-V5, SHARPIN-V5 with or without co-transfection of HOIL1, and subsequent α-HOIP immunoblotting. N =3. ( f ) HOIP protein level in B cells from the brother (+/ mut B) and patient ( mut / mut ) on PMA/Ionomycin stimulation over time. β-Actin, loading control, N =2. Orange arrowheads indicate decreased HOIP levels.

Techniques Used: Western Blot, Cotransfection, Plasmid Preparation, Immunoprecipitation, Transfection

MALT1 proteolysis of HOIL1 in human immortalized B cells reduces linear ubiquitination. ( a ) Immunoblot (lower gel) for linear ubiquitin conjugates in TUBE-enriched total ubiquitinated protein fraction from B cells from the brother (+/ mut B) and patient ( mut / mut ) after stimulation. Generation of C-HOIL1 by MALT1 was shown by α-HOIL1 immunoblot for reference (Pre-TUBE, upper gel). N =4. β-Actin, loading control. ( b ) Immunoblot for linear ubiquitin on the TUBE-enriched ubiquitinated protein fraction in B cells from the brother (+/ mut B) after PMA/ionomycin stimulation, with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =2. Immunoblotting for RIP1 a known substrate of LUBAC, was performed on the same samples. The number of ubiquitin moieties attached to each RIP1-polyubiquitinated species detected was calculated from their apparent molecular weights, and is indicated. NF-κB signalling was monitored by immunoblotting for IκBα and phospho-p65 (p-p65), N =6. β-Actin, loading control. ( c ) After initial stimulation by PMA/ionomycin for 15 min followed by cell washing, +/ mut B cells were again stimulated after washing at 120 min, designated 2 nd pulse 0 min. Cells were collected at the indicated times and lysates were probed for phospho-p65 and IκBα levels, N =6. β-Actin, loading control on the same blot as IκBα.
Figure Legend Snippet: MALT1 proteolysis of HOIL1 in human immortalized B cells reduces linear ubiquitination. ( a ) Immunoblot (lower gel) for linear ubiquitin conjugates in TUBE-enriched total ubiquitinated protein fraction from B cells from the brother (+/ mut B) and patient ( mut / mut ) after stimulation. Generation of C-HOIL1 by MALT1 was shown by α-HOIL1 immunoblot for reference (Pre-TUBE, upper gel). N =4. β-Actin, loading control. ( b ) Immunoblot for linear ubiquitin on the TUBE-enriched ubiquitinated protein fraction in B cells from the brother (+/ mut B) after PMA/ionomycin stimulation, with and without preincubation with MALT1 inhibitor z-VRPR-fmk. N =2. Immunoblotting for RIP1 a known substrate of LUBAC, was performed on the same samples. The number of ubiquitin moieties attached to each RIP1-polyubiquitinated species detected was calculated from their apparent molecular weights, and is indicated. NF-κB signalling was monitored by immunoblotting for IκBα and phospho-p65 (p-p65), N =6. β-Actin, loading control. ( c ) After initial stimulation by PMA/ionomycin for 15 min followed by cell washing, +/ mut B cells were again stimulated after washing at 120 min, designated 2 nd pulse 0 min. Cells were collected at the indicated times and lysates were probed for phospho-p65 and IκBα levels, N =6. β-Actin, loading control on the same blot as IκBα.

Techniques Used:

Defective NF-κB activation in MALT1 mut/mut B cells. ( a ) Simplified diagram showing the central role of the CARD11/BCL10/MALT1 (CBM) complex in B- and T-cell receptor controlled canonical NF-κB signalling pathway. ( b ) Family pedigree of the MALT1 genetic mutation. ( c ) Immunoblots of MALT1 before and after stimulation with PMA/ionomycin for 2 and 4 h in immortalized B cells from the MALT1-(Trp580Ser) homozygous daughter ( mut / mut ), the heterozygous brother (+/ mut B) and mother (+/ mut M), N =11. β-Actin, loading control. ( d ) NF-κB activation deficiency in B cells of patient ( mut / mut ) and mother (+/ mut M) after PMA/ionomycin stimulation was shown by IκBα degradation (left) and phosphorylation of the p65 subunit of NF-κB (p-p65; right), mean±s.d. Bonferroni post-test after two-way analysis of variance: * P
Figure Legend Snippet: Defective NF-κB activation in MALT1 mut/mut B cells. ( a ) Simplified diagram showing the central role of the CARD11/BCL10/MALT1 (CBM) complex in B- and T-cell receptor controlled canonical NF-κB signalling pathway. ( b ) Family pedigree of the MALT1 genetic mutation. ( c ) Immunoblots of MALT1 before and after stimulation with PMA/ionomycin for 2 and 4 h in immortalized B cells from the MALT1-(Trp580Ser) homozygous daughter ( mut / mut ), the heterozygous brother (+/ mut B) and mother (+/ mut M), N =11. β-Actin, loading control. ( d ) NF-κB activation deficiency in B cells of patient ( mut / mut ) and mother (+/ mut M) after PMA/ionomycin stimulation was shown by IκBα degradation (left) and phosphorylation of the p65 subunit of NF-κB (p-p65; right), mean±s.d. Bonferroni post-test after two-way analysis of variance: * P

Techniques Used: Activation Assay, Mutagenesis, Western Blot

Cleavage of HOIL1 by MALT1 leads to decreased linear ubiquitination in primary lymphocytes. ( a ) Immunoblotting for total linear ubiquitination and HOIL1 cleavage in the TUBE-enriched fraction from α-IgG/M-CD40L primed tonsil B-cell homogenates from a normal (+/+N) subject on PMA/ionomycin stimulation. * Represents nonspecific band. β-Actin, loading control. ( b ) TUBE pulldown and subsequent immunoblotting for linear ubiquitin conjugates and the known LUBAC target NEMO in tonsil B-cell homogenates from normal (+/+N) subjects on stimulation with PMA/ionomycin. Cells were primed with α-IgG/M and CD40L. NEMO-polyubiquitin conjugates were calculated as marked. N =7 for a and b . ( c ) α-N-HOIL1 immunoblot for PMA/ionomycin-induced cleavage of HOIL1 in Jurkat T cells with and without MALT1 inhibitors z-VRPR-fmk (20 μM) and Mepazine (20 μM). * Represents nonspecific bands. ( d ) α-HOIL1 immunoblot for HOIL1 cleavage on stimulation of expanded primary CD4 + T cells from the brother (+/ mut B) with PMA/ionomycin and with α-CD3/α-CD28 coated beads ( N =3). The polyclonal antibody detects both the C-terminal and the N-terminal cleavage products. * Represents consistently observed nonspecific band. β-Actin, loading control. ( e ) Immunoblots for total linear ubiquitination after TUBE pulldown in expanded primary CD4 + T cells from the brother (+/ mut B) on stimulation with PMA/ionomycin. K48-linked ubiquitination was included for reference. β-Actin, loading control. ( f ) Immunoblots for total linear ubiquitin conjugates and NEMO in PMA/ionomycin-stimulated expanded primary CD4 + T cells from the brother (+/ mut B), with and without pretreatment with MALT1 inhibitor z-VRPR-fmk ( N =3). NEMO-polyubiquitin conjugates were calculated as marked.
Figure Legend Snippet: Cleavage of HOIL1 by MALT1 leads to decreased linear ubiquitination in primary lymphocytes. ( a ) Immunoblotting for total linear ubiquitination and HOIL1 cleavage in the TUBE-enriched fraction from α-IgG/M-CD40L primed tonsil B-cell homogenates from a normal (+/+N) subject on PMA/ionomycin stimulation. * Represents nonspecific band. β-Actin, loading control. ( b ) TUBE pulldown and subsequent immunoblotting for linear ubiquitin conjugates and the known LUBAC target NEMO in tonsil B-cell homogenates from normal (+/+N) subjects on stimulation with PMA/ionomycin. Cells were primed with α-IgG/M and CD40L. NEMO-polyubiquitin conjugates were calculated as marked. N =7 for a and b . ( c ) α-N-HOIL1 immunoblot for PMA/ionomycin-induced cleavage of HOIL1 in Jurkat T cells with and without MALT1 inhibitors z-VRPR-fmk (20 μM) and Mepazine (20 μM). * Represents nonspecific bands. ( d ) α-HOIL1 immunoblot for HOIL1 cleavage on stimulation of expanded primary CD4 + T cells from the brother (+/ mut B) with PMA/ionomycin and with α-CD3/α-CD28 coated beads ( N =3). The polyclonal antibody detects both the C-terminal and the N-terminal cleavage products. * Represents consistently observed nonspecific band. β-Actin, loading control. ( e ) Immunoblots for total linear ubiquitination after TUBE pulldown in expanded primary CD4 + T cells from the brother (+/ mut B) on stimulation with PMA/ionomycin. K48-linked ubiquitination was included for reference. β-Actin, loading control. ( f ) Immunoblots for total linear ubiquitin conjugates and NEMO in PMA/ionomycin-stimulated expanded primary CD4 + T cells from the brother (+/ mut B), with and without pretreatment with MALT1 inhibitor z-VRPR-fmk ( N =3). NEMO-polyubiquitin conjugates were calculated as marked.

Techniques Used: Western Blot

26) Product Images from "Importance of Th22 Cell Disequilibrium in Immune Thrombocytopenic Purpura"

Article Title: Importance of Th22 Cell Disequilibrium in Immune Thrombocytopenic Purpura

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.912528

Quantification of circulating CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells in each group. ( A–C ) Representative four-color dot plot analyses of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells after stimulation with PMA and ionomycin. ( D ) Frequency of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group. Results are represented as the mean ±SD (** p
Figure Legend Snippet: Quantification of circulating CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells in each group. ( A–C ) Representative four-color dot plot analyses of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells after stimulation with PMA and ionomycin. ( D ) Frequency of CD4 + IFN-γ + , CD4 + IL-17A + , and IL-22 + cells on the gated lymphocytes in the FSC/SSC plot in each group. Results are represented as the mean ±SD (** p

Techniques Used:

27) Product Images from "Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells *"

Article Title: Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.364737

Effects of the caspase inhibitor Z-VAD-FMK on ionomycin and ABT-263-mediated cell death in MDA-MB-231 breast cancer cells. A–F , dot plots for control, ionomycin, or ABT-263 either in the absence or presence of Z-VAD-FMK. Each dot plot represents
Figure Legend Snippet: Effects of the caspase inhibitor Z-VAD-FMK on ionomycin and ABT-263-mediated cell death in MDA-MB-231 breast cancer cells. A–F , dot plots for control, ionomycin, or ABT-263 either in the absence or presence of Z-VAD-FMK. Each dot plot represents

Techniques Used: Multiple Displacement Amplification

PMCA1 and PMCA4 silencing effects in promoting ionomycin-mediated cell death in MDA-MB-231 breast cancer cells. Dot plots of Hoechst 33342 and propidium iodide fluorescence in cells transfected with siNT ( A and D ), siPMCA4 ( B and E ), or siPMCA1 ( C and
Figure Legend Snippet: PMCA1 and PMCA4 silencing effects in promoting ionomycin-mediated cell death in MDA-MB-231 breast cancer cells. Dot plots of Hoechst 33342 and propidium iodide fluorescence in cells transfected with siNT ( A and D ), siPMCA4 ( B and E ), or siPMCA1 ( C and

Techniques Used: Multiple Displacement Amplification, Fluorescence, Transfection

Ionomycin-evoked [Ca 2+ ] CYT signals in the presence of PMCA1 and PMCA4 silencing. MDA-MB-231 breast cancer cells were transfected with siPMCA1, siPMCA4, or siNT, and changes in [Ca 2+ ] CYT were assessed after the addition of ionomycin (3 μ m ( A ) or
Figure Legend Snippet: Ionomycin-evoked [Ca 2+ ] CYT signals in the presence of PMCA1 and PMCA4 silencing. MDA-MB-231 breast cancer cells were transfected with siPMCA1, siPMCA4, or siNT, and changes in [Ca 2+ ] CYT were assessed after the addition of ionomycin (3 μ m ( A ) or

Techniques Used: Multiple Displacement Amplification, Transfection

28) Product Images from "TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore"

Article Title: TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197622

The phosphorylation state of TRESK does not influence the kinetics of Ba 2+ block. Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K + -free bath solution to a high K + solution (solution changes are marked with bars above the graphs). Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. TRESK channels were either dephosphorylated by application of 0.5 μM ionomycin to the bath solution before recording or phosphorylated by application of purified MARK2 (16 μg/ml), 30 U/ml PKA (1 mM cAMP and 1 mM DTT was added to ensure the enzymatic activity of PKA) and 2 mM ATP before the initiation of the Ba 2+ block. A, Representative recording, TRESK channels were dephosphorylated before patch excision by application of 0.5 μM ionomycin to the bath solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, Representative recording, TRESK channels were phosphorylated by perfusing the intracellular side of the patch with a bath solution containing both kinases (purified MARK2, PKA, 1 mM cAMP, 1 mM DTT and 2 mM ATP) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). C, The kinetics of TRESK current inhibition by Ba 2+ were determined for both the phosphorylated and dephosphorylated channel (n = 6 and 5 patches). The average normalized curves for both groups are plotted. The inset shows the onset of Ba 2+ with a higher temporal resolution. D, The current recordings of the Ba 2+ block recorded for both the phosphorylated and dephosphorylated groups were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. The difference between the groups was not statistically significant (Student’s t test).
Figure Legend Snippet: The phosphorylation state of TRESK does not influence the kinetics of Ba 2+ block. Mouse TRESK channels were expressed in HEK293T cells. Experiments were done on excised inside-out patches. TRESK currents were measured at +60 mV by switching the K + -free bath solution to a high K + solution (solution changes are marked with bars above the graphs). Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV. TRESK channels were either dephosphorylated by application of 0.5 μM ionomycin to the bath solution before recording or phosphorylated by application of purified MARK2 (16 μg/ml), 30 U/ml PKA (1 mM cAMP and 1 mM DTT was added to ensure the enzymatic activity of PKA) and 2 mM ATP before the initiation of the Ba 2+ block. A, Representative recording, TRESK channels were dephosphorylated before patch excision by application of 0.5 μM ionomycin to the bath solution. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). B, Representative recording, TRESK channels were phosphorylated by perfusing the intracellular side of the patch with a bath solution containing both kinases (purified MARK2, PKA, 1 mM cAMP, 1 mM DTT and 2 mM ATP) as shown on the graph. Barium (1 mM) was applied at a holding potential of -80 mV and block was initiated by depolarizing the membrane to +60 mV (see the vertical arrow , application of Ba 2+ is marked by a bar above the recording and changes in the membrane potential are shown under the recording). C, The kinetics of TRESK current inhibition by Ba 2+ were determined for both the phosphorylated and dephosphorylated channel (n = 6 and 5 patches). The average normalized curves for both groups are plotted. The inset shows the onset of Ba 2+ with a higher temporal resolution. D, The current recordings of the Ba 2+ block recorded for both the phosphorylated and dephosphorylated groups were fitted with a double exponential equation. The time constants of the fitted equations are plotted as a scatter plot. The average values are plotted as columns. The difference between the groups was not statistically significant (Student’s t test).

Techniques Used: Blocking Assay, Purification, Activity Assay, Inhibition

29) Product Images from "Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease"

Article Title: Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease

Journal: Gastroenterology

doi: 10.1053/j.gastro.2018.02.019

Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; PMA/Ionomycin stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P
Figure Legend Snippet: Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; PMA/Ionomycin stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

Techniques Used: Isolation, Selection, Positive Control, FACS, Two Tailed Test, MANN-WHITNEY

Intrahepatic γδ T Cells Isolated from PSC Patient Livers are Capable of Producing IL-17. (A–B) Bulk liver lymphocytes from human subjects with ESLD as a result of PSC ( n=5 ) or HCV ( n=5) were stimulated overnight with PMA/Ionomycin as described. γδ T cells from PSC patients were capable of IL-17 production (A, left; * P
Figure Legend Snippet: Intrahepatic γδ T Cells Isolated from PSC Patient Livers are Capable of Producing IL-17. (A–B) Bulk liver lymphocytes from human subjects with ESLD as a result of PSC ( n=5 ) or HCV ( n=5) were stimulated overnight with PMA/Ionomycin as described. γδ T cells from PSC patients were capable of IL-17 production (A, left; * P

Techniques Used: Isolation

Fibrotic Liver γδ T Cell Compartment is Altered in Favor of IL-17 Producing Subsets. (A–B) Mdr2 −/− and FVB/N mice were administered 500μg of anti-γδTCR (Clone UC7-13D5) or appropriate Hamster-IgG isotype control intravenously and sacrificed one day following treatment. Livers and spleens were harvested; and bulk lymphocytes were first stained with anti-hamster IgG, followed by TCRVγ1.1–1.2, Vγ2, Vγ3, and for Vγ4, Vγ7 in combination with appropriate surface antibodies. Frequencies were determined based on the percentage of the indicated Vγ-chain within the live non-autofluorescent TCRβ- CD3+ gate (A–B). “Other” refers to in vivo bound and ex vivo detectable UC7-13D5 (A, top; B, Top). (C–D) Lymphocytes were stimulated with PMA/ionomycin in the presence of Golgi Plug/Golgi Stop for 4 hours at 37°C, and subsequently stained for intracellular IL-17A. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P
Figure Legend Snippet: Fibrotic Liver γδ T Cell Compartment is Altered in Favor of IL-17 Producing Subsets. (A–B) Mdr2 −/− and FVB/N mice were administered 500μg of anti-γδTCR (Clone UC7-13D5) or appropriate Hamster-IgG isotype control intravenously and sacrificed one day following treatment. Livers and spleens were harvested; and bulk lymphocytes were first stained with anti-hamster IgG, followed by TCRVγ1.1–1.2, Vγ2, Vγ3, and for Vγ4, Vγ7 in combination with appropriate surface antibodies. Frequencies were determined based on the percentage of the indicated Vγ-chain within the live non-autofluorescent TCRβ- CD3+ gate (A–B). “Other” refers to in vivo bound and ex vivo detectable UC7-13D5 (A, top; B, Top). (C–D) Lymphocytes were stimulated with PMA/ionomycin in the presence of Golgi Plug/Golgi Stop for 4 hours at 37°C, and subsequently stained for intracellular IL-17A. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

Techniques Used: Mouse Assay, Staining, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Intrahepatic γδ T Cells are Predominant Source of IL-17A During Cholestatic Liver Disease. (A) Liver tissues from 25-wk-old FVB/N and Mdr2 −/− mice were sectioned and stained with Sirius red and H E stain (Mag. 100×). (B) Serum samples were obtained from 25-wk-old Mdr2 −/− and FVB/N mice and the levels of IL-17A were detected using a standard ELISA. (C–D) Bulk lymphocytes isolated from livers and spleens were stimulated with PMA/Ionomycin for 4 hours to determine frequency of live, non-autofluorescent CD3+ (T cells) (C) that produce IL-17A from livers and spleens derived from the indicated group. (D) Quantitation of frequencies of IL-17A+ CD4+ T cells (Top) and γδ T cells (bottom) from livers are shown. Representative figures from more than 3 independent experiments are shown. Two-tailed Mann Whitney test, *** P
Figure Legend Snippet: Intrahepatic γδ T Cells are Predominant Source of IL-17A During Cholestatic Liver Disease. (A) Liver tissues from 25-wk-old FVB/N and Mdr2 −/− mice were sectioned and stained with Sirius red and H E stain (Mag. 100×). (B) Serum samples were obtained from 25-wk-old Mdr2 −/− and FVB/N mice and the levels of IL-17A were detected using a standard ELISA. (C–D) Bulk lymphocytes isolated from livers and spleens were stimulated with PMA/Ionomycin for 4 hours to determine frequency of live, non-autofluorescent CD3+ (T cells) (C) that produce IL-17A from livers and spleens derived from the indicated group. (D) Quantitation of frequencies of IL-17A+ CD4+ T cells (Top) and γδ T cells (bottom) from livers are shown. Representative figures from more than 3 independent experiments are shown. Two-tailed Mann Whitney test, *** P

Techniques Used: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Derivative Assay, Quantitation Assay, Two Tailed Test, MANN-WHITNEY

Related Articles

Flow Cytometry:

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells
Article Snippet: .. For PMA/ionomycin stimulation in vitro, splenocytes from control and Ncr1 Ki Ptpn11 fl/fl mice were left unstimulated or stimulated either 15 min with 12.5 nM PMA plus 0.125 µg/ml ionomycin or 2 h with 50 nM PMA plus 0.5 µg/ml ionomycin followed by 2 h with Brefeldin A (10 µg/mL Enzo Life Science) for the detection by flow cytometry of intracellular protein phosphorylation and cytokine production, respectively. .. In cytokine production experiments, anti-CD107α (LAMP-1) antibody was added at the beginning of incubation to detect NK cell degranulation.

In Vitro:

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells
Article Snippet: .. For PMA/ionomycin stimulation in vitro, splenocytes from control and Ncr1 Ki Ptpn11 fl/fl mice were left unstimulated or stimulated either 15 min with 12.5 nM PMA plus 0.125 µg/ml ionomycin or 2 h with 50 nM PMA plus 0.5 µg/ml ionomycin followed by 2 h with Brefeldin A (10 µg/mL Enzo Life Science) for the detection by flow cytometry of intracellular protein phosphorylation and cytokine production, respectively. .. In cytokine production experiments, anti-CD107α (LAMP-1) antibody was added at the beginning of incubation to detect NK cell degranulation.

Cytometry:

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells
Article Snippet: .. For PMA/ionomycin stimulation in vitro, splenocytes from control and Ncr1 Ki Ptpn11 fl/fl mice were left unstimulated or stimulated either 15 min with 12.5 nM PMA plus 0.125 µg/ml ionomycin or 2 h with 50 nM PMA plus 0.5 µg/ml ionomycin followed by 2 h with Brefeldin A (10 µg/mL Enzo Life Science) for the detection by flow cytometry of intracellular protein phosphorylation and cytokine production, respectively. .. In cytokine production experiments, anti-CD107α (LAMP-1) antibody was added at the beginning of incubation to detect NK cell degranulation.

Mouse Assay:

Article Title: Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells
Article Snippet: .. For PMA/ionomycin stimulation in vitro, splenocytes from control and Ncr1 Ki Ptpn11 fl/fl mice were left unstimulated or stimulated either 15 min with 12.5 nM PMA plus 0.125 µg/ml ionomycin or 2 h with 50 nM PMA plus 0.5 µg/ml ionomycin followed by 2 h with Brefeldin A (10 µg/mL Enzo Life Science) for the detection by flow cytometry of intracellular protein phosphorylation and cytokine production, respectively. .. In cytokine production experiments, anti-CD107α (LAMP-1) antibody was added at the beginning of incubation to detect NK cell degranulation.

Incubation:

Article Title: Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome
Article Snippet: .. Briefly, heparinized peripheral whole blood (400 µl) with an equal volume of Roswell Park Memorial Institute 1640 medium was incubated for 4h at 37°C, 5% CO2 in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 µg/ml of ionomycin, and 1.7 µg/ml Golgiplug (monensin; all from Alexis Biochemicals, San Diego, CA, USA). .. PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity.

Article Title: Elevated expression of interleukin-21 and its correlation to T-cell subpopulation in patients with ulcerative colitis
Article Snippet: .. Briefly, heparinized peripheral whole blood (400 µl) with an equal volume of RPMI 1640 medium was incubated for 4 h at 37°C, 5% CO2 in the presence of 25 ng/ml of phorbol myristate acetate (PMA), 1 µg/ml of ionomycin, and 1.7 µg/ml GolgiPlug (Monensin) (all from Alexis Biochemicals, San Diego, CA). .. Phorbol myristate acetate and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the TCR complex and have the advantage of stimulating T cells of any antigen specificity.

Article Title: Increased Number of Tc17 and Correlation with Th17 Cells in Patients with Immune Thrombocytopenia
Article Snippet: .. Briefly, heparinized peripheral whole blood (400 µl) with an equal volume of Roswell Park Memorial Institute (RPMI)-1640 medium was incubated for 4 h at 37°C in 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 µg/mL of ionomycin, and 1.7 µg/mL of monensin (all from Alexis Biochemicals, San Diego, CA, USA). .. PMA and ionomycin are pharmacologic T cell-activating agents that mimic signals generated by the T cell receptor (TCR) complex and have the advantage of stimulating T cells of any Ag specificity.

Article Title: Increased Frequencies of Th22 Cells as well as Th17 Cells in the Peripheral Blood of Patients with Ankylosing Spondylitis and Rheumatoid Arthritis
Article Snippet: .. Briefly, heparinized peripheral whole blood (400 µl) with an equal volume of Roswell Park Memorial Institute 1640 medium were incubated for 4 h at 37°C, 5% CO2 in the presence of 25 ng/mL of phorbol myristate acetate (PMA), 1 µg/mL of ionomycin, and 1.7 µg/ml Golgiplug(Monensin; all from Alexis Biochemicals, San Diego, CA, USA). .. PMA and ionomycin are pharmacological T-cell-activating agents that mimic signals generated by the T-cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity.

other:

Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.
Article Snippet: Ionomycin was purchased from Alexis Biochemicals (San Diego, CA).

Article Title: Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells [S]
Article Snippet: Further, lyophilized 2-chloro-5-nitro- N -4-pyridinylbenzamide (T0070907), phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A (all Enzo, Lörrach, Germany), lipopolysaccharide (LPS from E.coli , Serotype 0111:B4, Sigma-Aldrich, Taufkirchen, Germany), and concanavalin A (ConA, Sigma-Aldrich) were solubilized in DMSO, aliquoted, and stored at −20°C.

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    Enzo Biochem ionomycin
    Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and <t>ionomycin</t> for subsequent 5 h. Th cells were identified
    Ionomycin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and ionomycin for subsequent 5 h. Th cells were identified

    Journal: Journal of Lipid Research

    Article Title: Evaluation of suppressive and pro-resolving effects of EPA and DHA in human primary monocytes and T-helper cells [S]

    doi: 10.1194/jlr.P031260

    Figure Lengend Snippet: Effect of EPA and DHA on cytokine production of Th cells (CD3 + CD4 + ). (A) PBMC from buffy coats were incubated without or with increasing concentrations of EPA or DHA for 19 h and activated by PMA and ionomycin for subsequent 5 h. Th cells were identified

    Article Snippet: Further, lyophilized 2-chloro-5-nitro- N -4-pyridinylbenzamide (T0070907), phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A (all Enzo, Lörrach, Germany), lipopolysaccharide (LPS from E.coli , Serotype 0111:B4, Sigma-Aldrich, Taufkirchen, Germany), and concanavalin A (ConA, Sigma-Aldrich) were solubilized in DMSO, aliquoted, and stored at −20°C.

    Techniques: Incubation

    WGP induces enhanced CTL priming in vivo . Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in Materials and Methods . (A, B) Single cell suspensions prepared from spleens (A) and draining lymph nodes (B) were stimulated with PMA plus ionomycin and stained intracellular IFN-γ. Cells were gated on CD3 + CD8 + T cells. Culture supernatants from splenocytes and lymphoid cells were collected and assayed for IFN-γ using ELISA. (C) Tumor specimens from each group were prepared for single cell suspensions. Cells were stained with mAbs against CD3, CD8 and were assessed by flow cytometry. Cells were gated on CD3 + T cells. RNAs from tumor specimens were extracted and qRT-PCR was performed for IFN-γ. Results are expressed as mean ± SD. **P

    Journal: PLoS ONE

    Article Title: Up-Regulation of GITRL on Dendritic Cells by WGP Improves Anti-Tumor Immunity in Murine Lewis Lung Carcinoma

    doi: 10.1371/journal.pone.0046936

    Figure Lengend Snippet: WGP induces enhanced CTL priming in vivo . Groups of mice (n = 6) bearing established Lewis lung carcinoma were treated as described in Materials and Methods . (A, B) Single cell suspensions prepared from spleens (A) and draining lymph nodes (B) were stimulated with PMA plus ionomycin and stained intracellular IFN-γ. Cells were gated on CD3 + CD8 + T cells. Culture supernatants from splenocytes and lymphoid cells were collected and assayed for IFN-γ using ELISA. (C) Tumor specimens from each group were prepared for single cell suspensions. Cells were stained with mAbs against CD3, CD8 and were assessed by flow cytometry. Cells were gated on CD3 + T cells. RNAs from tumor specimens were extracted and qRT-PCR was performed for IFN-γ. Results are expressed as mean ± SD. **P

    Article Snippet: For intracellular cytokine staining, single cell suspensions were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 µg/ml), monensin (Enzo, 2 µg/ml).

    Techniques: CTL Assay, In Vivo, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR