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  • 99
    Thermo Fisher phorbol 12 myristate 13 acetate pma
    Phorbol 12 Myristate 13 Acetate Pma, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pma
    Cytotoxicity of the capsule-deficient A20 and AP3 (SCN162 and SCN157, respectively), SpeB C192S mutant of SCN162 (SCN208), and slo mutant of SCN157 (SCN176) on <t>PMA-activated</t> <t>U937</t> cells with or without cytochalasin D during infection. (A,B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells after 0.5 h (T 0 ) and 0.75 h (T 0.75 ) of infection. The right panel shows the infection protocol of (A,B,D,E) . (C) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D (CytoD) treatment. The lower panel shows the infection protocol. (D,E) Cytotoxicity of SCN162, SCN157, SCN176, and SCN208 on PMA-activated U937 cells. Error bars represent the standard deviations. ∗ p
    Pma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phorbol 12 myristate 13 acetate pma
    Cytotoxicity of the capsule-deficient A20 and AP3 (SCN162 and SCN157, respectively), SpeB C192S mutant of SCN162 (SCN208), and slo mutant of SCN157 (SCN176) on <t>PMA-activated</t> <t>U937</t> cells with or without cytochalasin D during infection. (A,B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells after 0.5 h (T 0 ) and 0.75 h (T 0.75 ) of infection. The right panel shows the infection protocol of (A,B,D,E) . (C) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D (CytoD) treatment. The lower panel shows the infection protocol. (D,E) Cytotoxicity of SCN162, SCN157, SCN176, and SCN208 on PMA-activated U937 cells. Error bars represent the standard deviations. ∗ p
    Phorbol 12 Myristate 13 Acetate Pma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phorbol myristate acetate pma
    Cytotoxicity of the capsule-deficient A20 and AP3 (SCN162 and SCN157, respectively), SpeB C192S mutant of SCN162 (SCN208), and slo mutant of SCN157 (SCN176) on <t>PMA-activated</t> <t>U937</t> cells with or without cytochalasin D during infection. (A,B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells after 0.5 h (T 0 ) and 0.75 h (T 0.75 ) of infection. The right panel shows the infection protocol of (A,B,D,E) . (C) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D (CytoD) treatment. The lower panel shows the infection protocol. (D,E) Cytotoxicity of SCN162, SCN157, SCN176, and SCN208 on PMA-activated U937 cells. Error bars represent the standard deviations. ∗ p
    Phorbol Myristate Acetate Pma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pma  (Biotium)
    92
    Biotium pma
    PRRSV and host genomic detection in spiked tissues following <t>EMA</t> or <t>PMA</t> treatment using qPCR. Effect of EMA and PMA treatments, with or without ultracentrifugation, on (A) PRRSV quantification and (B) host genomic DNA (β-Actin) in lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL or 50,000 TCID 50 /mL) or in serum spiked with PRRSV (5000 TCID 50 /mL). Results are expressed as Ct and were obtained from two to seven independent experiments. The results of each independent experiment (trial) are illustrated in Supplemental Fig. 1. Sample (TCID 50 /mL) represents the type of tissue spiked with PRRSV. Numbers in brackets represent the PRRSV concentration for each spiked sample expressed in TCID 50 /mL. Open bars represent results obtained from samples processed without an ultracentrifugation step while filled bars represent results obtained from samples treated with an ultracentrifugation step. A Ct value of 37 (dashed line) represents the limit of detection of each qPCR test. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P
    Pma, supplied by Biotium, used in various techniques. Bioz Stars score: 92/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pma
    Cytokines production by reactivated splenocytes from mice treated with recombinant bacteria and challenged with TC-1 cells. Splenocytes were stimulated for 48 h with <t>PMA</t> <t>ionomycin</t> before measuring cytokines levels. Data represented mean ± SEM from 4 ( L. lactis wt) to 8 mice ( L. lactis IL-17). Data are analyzed with unpaired t -test followed by Mann–Withney post-test.
    Pma, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 688 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pma ionomycin
    CD39+CD103+ T RM cells sorted from human endometrial tumors are transcriptionally active. ( A ) Overview of experimental set-up. CD39+CD103+ T RM cells and CD8+ TIL were sorted from human high-grade endometrial cancer digests ( n = 3). Subsequently, cells remained untreated or were treated for 4.5 h with actinomycin D, or 4 h of <t>PMA/ionomyin</t> or pre-incubation for 30 min with actinomycin D followed by 4 h <t>PMA/ionomycin.</t> ( B ) Concentration of cDNA (ng/μL/100 cells) of CD39+CD103+ T RM cells and CD8+ TIL per treatment group. The median concentration + 95% confidence interval is depicted. ( C ) Principal component analysis of mRNA sequencing data of all T RM samples. The first two principal components are depicted. Individual patient samples are identified by separate colors. ( D ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of untreated CD39+CD103+ T RM cells from three patients. The scale applies to all similar heatmaps of CD39+CD103+ T RM cells throughout the paper.
    Pma Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris phorbol 12 myristate 13 acetate pma
    CD39+CD103+ T RM cells sorted from human endometrial tumors are transcriptionally active. ( A ) Overview of experimental set-up. CD39+CD103+ T RM cells and CD8+ TIL were sorted from human high-grade endometrial cancer digests ( n = 3). Subsequently, cells remained untreated or were treated for 4.5 h with actinomycin D, or 4 h of <t>PMA/ionomyin</t> or pre-incubation for 30 min with actinomycin D followed by 4 h <t>PMA/ionomycin.</t> ( B ) Concentration of cDNA (ng/μL/100 cells) of CD39+CD103+ T RM cells and CD8+ TIL per treatment group. The median concentration + 95% confidence interval is depicted. ( C ) Principal component analysis of mRNA sequencing data of all T RM samples. The first two principal components are depicted. Individual patient samples are identified by separate colors. ( D ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of untreated CD39+CD103+ T RM cells from three patients. The scale applies to all similar heatmaps of CD39+CD103+ T RM cells throughout the paper.
    Phorbol 12 Myristate 13 Acetate Pma, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    LC Laboratories pma
    SynGAP Regulates ERK Signaling to Control Excitatory Synaptic Strength. (A) Example images and the normalized mean (+SEM) intensity of AHA signals are presented from experiments using FUNCAT to measure protein synthesis rates. SynGAPsiRNA-mediated increase in AHA-incorporation rates was suppressed by treatment with the ERK blocker <t>U0126</t> (25 µM, 6 hr). Scale bar = 5 µm. (B) Knockdown of SynGAP also increased levels of phosphorylated P70S6K, and this increase was suppressed by U0126. (C) U0126 rescued the increase in mEPSC amplitudes in neurons transfected with SynGAPsiRNA to control levels. The <t>PMA-induced</t> increase in mEPSC amplitudes in control neurons was occluded in neurons expressing SynGAPsiRNA. The increase in mEPSC amplitudes in cells expressing SynGAPsiRNA was not affected by the PKA blocker PKI 14–22 (1 µM, 6 hr). Note: the GFP control data in (C), shown for comparison, are also presented in Figure 2C .
    Pma, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam phorbol 12 myristate 13 acetate pma
    SynGAP Regulates ERK Signaling to Control Excitatory Synaptic Strength. (A) Example images and the normalized mean (+SEM) intensity of AHA signals are presented from experiments using FUNCAT to measure protein synthesis rates. SynGAPsiRNA-mediated increase in AHA-incorporation rates was suppressed by treatment with the ERK blocker <t>U0126</t> (25 µM, 6 hr). Scale bar = 5 µm. (B) Knockdown of SynGAP also increased levels of phosphorylated P70S6K, and this increase was suppressed by U0126. (C) U0126 rescued the increase in mEPSC amplitudes in neurons transfected with SynGAPsiRNA to control levels. The <t>PMA-induced</t> increase in mEPSC amplitudes in control neurons was occluded in neurons expressing SynGAPsiRNA. The increase in mEPSC amplitudes in cells expressing SynGAPsiRNA was not affected by the PKA blocker PKI 14–22 (1 µM, 6 hr). Note: the GFP control data in (C), shown for comparison, are also presented in Figure 2C .
    Phorbol 12 Myristate 13 Acetate Pma, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pma ionomycin
    Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with <t>PMA+ionomycin.</t> In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P
    Pma Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM phorbol 12 myristate 13 acetate pma
    Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), <t>phorbol-12-myristate-13-acetate</t> (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P
    Phorbol 12 Myristate 13 Acetate Pma, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotium propidium monoazide pma
    Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), <t>phorbol-12-myristate-13-acetate</t> (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P
    Propidium Monoazide Pma, supplied by Biotium, used in various techniques. Bioz Stars score: 88/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson phorbol 12 myristate 13 acetate pma
    Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), <t>phorbol-12-myristate-13-acetate</t> (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P
    Phorbol 12 Myristate 13 Acetate Pma, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson phorbol myristate acetate pma
    Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), <t>phorbol-12-myristate-13-acetate</t> (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P
    Phorbol Myristate Acetate Pma, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen pma
    Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with <t>DMSO,</t> 5 µM nutlin, <t>PMA,</t> or nutlin + PMA. Statistically significant changes by Student’s t test ( P
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    Cayman Chemical pma
    Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with <t>DMSO,</t> 5 µM nutlin, <t>PMA,</t> or nutlin + PMA. Statistically significant changes by Student’s t test ( P
    Pma, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 94/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA phorbol 12 myristate 13 acetate pma
    Identification of substrates specific for RHBDL2 ( A ). Strep tagged substrates were co-expressed with HA tagged forms of mouse RHBDL2, the four human rhomboids (R1/R2/R3/R4) and their corresponding inactivated forms where the catalytic serine residue was mutated to an alanine (SA). Twenty four hours after transfection, the medium was replaced by serum-free medium containing 10 µM broad spectrum matrix metalloprotease inhibitor BB94 to exclude shedding by these extracellular proteases. Forty eight hours after transfection, the media and cell lysates were harvested and analysed by immunoblotting. Cells were co-transfected with FLAG tagged prolactin as a secretion control and to confirm recovery of TCA precipitated proteins from the media. ( B ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 1 µM ionomycin, 1 µM ionomycin and 2 mM EGTA, or 1 µM ionomycin and 10 µM BB94. One hour after media replacement the media (upper panels) and cell lysate (lower panels) were harvested as previously described and analysed by immunoblotting. ( C ) Twenty four hours after transfection with plasmids encoding Strep tagged substrates, the medium was replaced with serum-free medium containing 0.5 µM phorbol 12-myristate 13-acetate <t>(PMA),</t> the inactive analogue 4α-phorbol 12,13-didecanoate (4αPDD), or both 0.5 µM PMA and 10 µM BB94. One hour after media replacement the media and cell lysate were harvested as previously described and analysed by immunoblotting. Asterisks indicate samples which required treatment with PNGase F prior to SDS-PAGE to reduce smearing and improve resolution of bands on the gel.
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    92
    Biomol GmbH pma
    Ser1134 and Ser1161 are the <t>PMA-</t> and <t>EGF-induced</t> SOS1 phosphorylation sites that conform to the minimal RSK consensus motif. ( A ) HEK 293 cells were treated as described in and whole cell extracts as well as ten percent of each immune complex
    Pma, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Electron Probe Microanalyzer
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    Ambient Carbon Monoxide Monitor
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    500 µg protein in 200 µl SDS PAGE Western blotting buffer human whole cell lysate epitheliod carcinoma cells induced with PMA whole cell lysate provided as Western blotting positive control
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    Image Search Results


    Cytotoxicity of the capsule-deficient A20 and AP3 (SCN162 and SCN157, respectively), SpeB C192S mutant of SCN162 (SCN208), and slo mutant of SCN157 (SCN176) on PMA-activated U937 cells with or without cytochalasin D during infection. (A,B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells after 0.5 h (T 0 ) and 0.75 h (T 0.75 ) of infection. The right panel shows the infection protocol of (A,B,D,E) . (C) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D (CytoD) treatment. The lower panel shows the infection protocol. (D,E) Cytotoxicity of SCN162, SCN157, SCN176, and SCN208 on PMA-activated U937 cells. Error bars represent the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells

    doi: 10.3389/fmicb.2018.02592

    Figure Lengend Snippet: Cytotoxicity of the capsule-deficient A20 and AP3 (SCN162 and SCN157, respectively), SpeB C192S mutant of SCN162 (SCN208), and slo mutant of SCN157 (SCN176) on PMA-activated U937 cells with or without cytochalasin D during infection. (A,B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells after 0.5 h (T 0 ) and 0.75 h (T 0.75 ) of infection. The right panel shows the infection protocol of (A,B,D,E) . (C) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D (CytoD) treatment. The lower panel shows the infection protocol. (D,E) Cytotoxicity of SCN162, SCN157, SCN176, and SCN208 on PMA-activated U937 cells. Error bars represent the standard deviations. ∗ p

    Article Snippet: In the present study, U937 cells were treated FCS-containing RPMI medium with 15 nM of PMA (1.5 mM in DMSO; SI-D2650, Sigma-Aldrich, St. Louis, MO, United States) for 3 days to induce cell differentiation from monocyte-like to macrophage-like cells.

    Techniques: Mutagenesis, Infection

    Survival fitness of the capsule-deficient A20 and AP3 in the culture broth and PMA-activated U937 cells. (A) Survival fitness of the capsule-deficient A20 (SCN156 [Cm S ] and SCN162 [Cm R ]) and AP3 (SCN157 [Cm S ] and SCN158 [Cm R ]) in trypticase soy broth with yeast extract (TSBY). An equal number of A20- and AP3-derivative strains was mixed and inoculated in TSBY. After 1.25–4.25 h of incubation, the number of A20 and AP3 mutants was determined using plating method. (B,D) Survival fitness of the capsule-deficient A20 (SCN162 [Cm R ]) and AP3 (SCN157 [Cm S ]) in PMA-activated U937 cells at multiplicity of infection (MOI) of 50 and 5 conditions. PMA-activated U937 cells were infected with mixed SCN162 and SCN157. The infection protocol is shown in the right panel. (C,E) The proportion of SCN157 and SCN162 in the recovered bacterial population after cells were infected with mixed SCN162 and SCN157 at MOI of 50 and 5 conditions. Cm: chloramphenicol. Error bars represent the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells

    doi: 10.3389/fmicb.2018.02592

    Figure Lengend Snippet: Survival fitness of the capsule-deficient A20 and AP3 in the culture broth and PMA-activated U937 cells. (A) Survival fitness of the capsule-deficient A20 (SCN156 [Cm S ] and SCN162 [Cm R ]) and AP3 (SCN157 [Cm S ] and SCN158 [Cm R ]) in trypticase soy broth with yeast extract (TSBY). An equal number of A20- and AP3-derivative strains was mixed and inoculated in TSBY. After 1.25–4.25 h of incubation, the number of A20 and AP3 mutants was determined using plating method. (B,D) Survival fitness of the capsule-deficient A20 (SCN162 [Cm R ]) and AP3 (SCN157 [Cm S ]) in PMA-activated U937 cells at multiplicity of infection (MOI) of 50 and 5 conditions. PMA-activated U937 cells were infected with mixed SCN162 and SCN157. The infection protocol is shown in the right panel. (C,E) The proportion of SCN157 and SCN162 in the recovered bacterial population after cells were infected with mixed SCN162 and SCN157 at MOI of 50 and 5 conditions. Cm: chloramphenicol. Error bars represent the standard deviations. ∗ p

    Article Snippet: In the present study, U937 cells were treated FCS-containing RPMI medium with 15 nM of PMA (1.5 mM in DMSO; SI-D2650, Sigma-Aldrich, St. Louis, MO, United States) for 3 days to induce cell differentiation from monocyte-like to macrophage-like cells.

    Techniques: Incubation, Infection

    Cytotoxicity of the capsule-deficient A20 (SCN162) and AP3 (SCN157) on PMA-activated U937 cells with or without penicillin, clindamycin, and cytochalasin D treatment after infection. (A) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without antibiotic treatment after 3.75 h of infection. (B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D or antibiotic treatments after 3.75 h of infection. The right panels of (A,B) show the infection protocols. Error bars represent the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells

    doi: 10.3389/fmicb.2018.02592

    Figure Lengend Snippet: Cytotoxicity of the capsule-deficient A20 (SCN162) and AP3 (SCN157) on PMA-activated U937 cells with or without penicillin, clindamycin, and cytochalasin D treatment after infection. (A) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without antibiotic treatment after 3.75 h of infection. (B) Cytotoxicity of SCN162 and SCN157 on PMA-activated U937 cells with or without cytochalasin D or antibiotic treatments after 3.75 h of infection. The right panels of (A,B) show the infection protocols. Error bars represent the standard deviations. ∗ p

    Article Snippet: In the present study, U937 cells were treated FCS-containing RPMI medium with 15 nM of PMA (1.5 mM in DMSO; SI-D2650, Sigma-Aldrich, St. Louis, MO, United States) for 3 days to induce cell differentiation from monocyte-like to macrophage-like cells.

    Techniques: Infection

    Survival fitness of the capsule-deficient A20 (SCN156), AP3 (SCN157), and slo mutants of SCN157 (SCN176) on PMA-activated U937 cells. PMA-activated U937 cells were infected with mixed (1:1 ratio) SCN156 and SCN176 at multiplicity of infection (MOI) of 50 or 5 as shown in panel (A) . (B,D) Survival fitness of SCN156 (Cm R ) and SCN176 (Cm S ) at MOI of 50 or 5 in gentamicin-free incubation conditions. (C,E) The proportion of SCN156 and SCN176 in the recovered bacterial population at MOI of 50 or 5 in gentamicin-free incubation conditions. Error bars represent the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells

    doi: 10.3389/fmicb.2018.02592

    Figure Lengend Snippet: Survival fitness of the capsule-deficient A20 (SCN156), AP3 (SCN157), and slo mutants of SCN157 (SCN176) on PMA-activated U937 cells. PMA-activated U937 cells were infected with mixed (1:1 ratio) SCN156 and SCN176 at multiplicity of infection (MOI) of 50 or 5 as shown in panel (A) . (B,D) Survival fitness of SCN156 (Cm R ) and SCN176 (Cm S ) at MOI of 50 or 5 in gentamicin-free incubation conditions. (C,E) The proportion of SCN156 and SCN176 in the recovered bacterial population at MOI of 50 or 5 in gentamicin-free incubation conditions. Error bars represent the standard deviations. ∗ p

    Article Snippet: In the present study, U937 cells were treated FCS-containing RPMI medium with 15 nM of PMA (1.5 mM in DMSO; SI-D2650, Sigma-Aldrich, St. Louis, MO, United States) for 3 days to induce cell differentiation from monocyte-like to macrophage-like cells.

    Techniques: Infection, Incubation

    Cell-association and intracellular survival amount of wild-type A20 strain, animal-passage covS mutant AP3, and their capsule-deficient mutants (SCN156 and SCN157, respectively) in PMA-activated U937 cells. (A,D) Cell-association amount of A20, AP3, SCN156, and SCN157 with U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized GAS strains at multiplicity of infection (MOI) of 50 or 0.5 for 30 min. The infected cells were washed after infection, and the number of cell-associated bacteria was determined using plating method. (B,E) Intracellular survival amount of A20, AP3, SCN156, and SCN157 with U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized GAS strains at MOI of 50 or 0.5 for 30 min. The infected cells were washed after infection and incubated in gentamicin-containing medium for 45 min. After gentamicin treatment, the infected cells were washed, and the number of intracellular bacteria was determined using plating method. (C,F) Cytotoxicity of A20, AP3, SCN156, and SCN157 to U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized bacteria at MOI of 50 or 0.5 for 45 min, and culture supernatants were collected for evaluating the cytotoxicity by lactic dehydrogenase (LDH) assay. Error bars represent the standard deviations. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Cytotoxicity and Survival Fitness of Invasive covS Mutant of Group A Streptococcus in Phagocytic Cells

    doi: 10.3389/fmicb.2018.02592

    Figure Lengend Snippet: Cell-association and intracellular survival amount of wild-type A20 strain, animal-passage covS mutant AP3, and their capsule-deficient mutants (SCN156 and SCN157, respectively) in PMA-activated U937 cells. (A,D) Cell-association amount of A20, AP3, SCN156, and SCN157 with U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized GAS strains at multiplicity of infection (MOI) of 50 or 0.5 for 30 min. The infected cells were washed after infection, and the number of cell-associated bacteria was determined using plating method. (B,E) Intracellular survival amount of A20, AP3, SCN156, and SCN157 with U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized GAS strains at MOI of 50 or 0.5 for 30 min. The infected cells were washed after infection and incubated in gentamicin-containing medium for 45 min. After gentamicin treatment, the infected cells were washed, and the number of intracellular bacteria was determined using plating method. (C,F) Cytotoxicity of A20, AP3, SCN156, and SCN157 to U937 cells. PMA-activated U937 cells were infected with the IgG-opsonized bacteria at MOI of 50 or 0.5 for 45 min, and culture supernatants were collected for evaluating the cytotoxicity by lactic dehydrogenase (LDH) assay. Error bars represent the standard deviations. ∗ p

    Article Snippet: In the present study, U937 cells were treated FCS-containing RPMI medium with 15 nM of PMA (1.5 mM in DMSO; SI-D2650, Sigma-Aldrich, St. Louis, MO, United States) for 3 days to induce cell differentiation from monocyte-like to macrophage-like cells.

    Techniques: Mutagenesis, Infection, Incubation, Lactate Dehydrogenase Assay

    PRRSV and host genomic detection in spiked tissues following EMA or PMA treatment using qPCR. Effect of EMA and PMA treatments, with or without ultracentrifugation, on (A) PRRSV quantification and (B) host genomic DNA (β-Actin) in lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL or 50,000 TCID 50 /mL) or in serum spiked with PRRSV (5000 TCID 50 /mL). Results are expressed as Ct and were obtained from two to seven independent experiments. The results of each independent experiment (trial) are illustrated in Supplemental Fig. 1. Sample (TCID 50 /mL) represents the type of tissue spiked with PRRSV. Numbers in brackets represent the PRRSV concentration for each spiked sample expressed in TCID 50 /mL. Open bars represent results obtained from samples processed without an ultracentrifugation step while filled bars represent results obtained from samples treated with an ultracentrifugation step. A Ct value of 37 (dashed line) represents the limit of detection of each qPCR test. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Journal: Journal of Virological Methods

    Article Title: Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification

    doi: 10.1016/j.jviromet.2015.06.014

    Figure Lengend Snippet: PRRSV and host genomic detection in spiked tissues following EMA or PMA treatment using qPCR. Effect of EMA and PMA treatments, with or without ultracentrifugation, on (A) PRRSV quantification and (B) host genomic DNA (β-Actin) in lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL or 50,000 TCID 50 /mL) or in serum spiked with PRRSV (5000 TCID 50 /mL). Results are expressed as Ct and were obtained from two to seven independent experiments. The results of each independent experiment (trial) are illustrated in Supplemental Fig. 1. Sample (TCID 50 /mL) represents the type of tissue spiked with PRRSV. Numbers in brackets represent the PRRSV concentration for each spiked sample expressed in TCID 50 /mL. Open bars represent results obtained from samples processed without an ultracentrifugation step while filled bars represent results obtained from samples treated with an ultracentrifugation step. A Ct value of 37 (dashed line) represents the limit of detection of each qPCR test. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Article Snippet: 2.3.2 Ethidium bromide monoazide and propidium monoazide treatments EMA and PMA (Biotium, Hayward, CA) were reconstituted according to manufacturer's recommendation.

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Labeling

    PRRSV detection in spiked samples by DNA array following EMA or PMA treatments. DNA array probes relative intensity from A) lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL), B) lung tissue homogenates spiked with PRRSV (50,000 TCID 50 /mL) and C) serum samples spiked with PRRSV (5000 TCID 50 /mL). Dots are relative fluorescence intensity mean values of two identical probes gathered from two to seven independent experiments (each experiment consisting of a duplicate of 34 PRRSV specific probes) and was calculated as followed: [(PFL − BFL)/BFL] where PFL represents a PRRSV probe fluorescence intensity and BFL represents the basal fluorescence level (negative control probe fluorescence). The results of each independent experiment (trial) are illustrated in Supplemental Fig. 2. The line represents the fluorescence mean value of all probes. Open dot circles represent results obtained from samples processed without an ultracentrifugation step while filled dot circles represent results obtained from samples treated with an ultracentrifugation step. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Journal: Journal of Virological Methods

    Article Title: Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification

    doi: 10.1016/j.jviromet.2015.06.014

    Figure Lengend Snippet: PRRSV detection in spiked samples by DNA array following EMA or PMA treatments. DNA array probes relative intensity from A) lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL), B) lung tissue homogenates spiked with PRRSV (50,000 TCID 50 /mL) and C) serum samples spiked with PRRSV (5000 TCID 50 /mL). Dots are relative fluorescence intensity mean values of two identical probes gathered from two to seven independent experiments (each experiment consisting of a duplicate of 34 PRRSV specific probes) and was calculated as followed: [(PFL − BFL)/BFL] where PFL represents a PRRSV probe fluorescence intensity and BFL represents the basal fluorescence level (negative control probe fluorescence). The results of each independent experiment (trial) are illustrated in Supplemental Fig. 2. The line represents the fluorescence mean value of all probes. Open dot circles represent results obtained from samples processed without an ultracentrifugation step while filled dot circles represent results obtained from samples treated with an ultracentrifugation step. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Article Snippet: 2.3.2 Ethidium bromide monoazide and propidium monoazide treatments EMA and PMA (Biotium, Hayward, CA) were reconstituted according to manufacturer's recommendation.

    Techniques: DNA Array, Fluorescence, Negative Control, Labeling

    PRRSV and host genomic detection efficiency in spiked tissue samples following EMA or PMA treatment by high-throughput sequencing. HTS results gathered from (A–C) lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL); from (D–F) lung tissue homogenates spiked with PRRSV (50,000 TCID 50 /mL); and from (G–I) serum samples spiked with PRRSV (5000 TCID 50 /mL). The amounts of PRRSV specific reads compared to the total number of reads gathered from each HTS run (expressed as %) are reported in panels (A), (D) and (G) while the percentage coverage of PRRSV recovered from the total number of PRRSV specific reads are reported in panel (B), (E) and (H). The host genomic specific reads compared to the total number of reads gathered from each HTS run (expressed as %) are reported in panels (C), (F) and (I). Open bars represent results obtained from samples processed without an ultracentrifugation step while filled bars represent results obtained from samples treated with an ultracentrifugation step. The results from each experiment are expressed separately in each graphic. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Journal: Journal of Virological Methods

    Article Title: Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification

    doi: 10.1016/j.jviromet.2015.06.014

    Figure Lengend Snippet: PRRSV and host genomic detection efficiency in spiked tissue samples following EMA or PMA treatment by high-throughput sequencing. HTS results gathered from (A–C) lung tissue homogenates spiked with PRRSV (5000 TCID 50 /mL); from (D–F) lung tissue homogenates spiked with PRRSV (50,000 TCID 50 /mL); and from (G–I) serum samples spiked with PRRSV (5000 TCID 50 /mL). The amounts of PRRSV specific reads compared to the total number of reads gathered from each HTS run (expressed as %) are reported in panels (A), (D) and (G) while the percentage coverage of PRRSV recovered from the total number of PRRSV specific reads are reported in panel (B), (E) and (H). The host genomic specific reads compared to the total number of reads gathered from each HTS run (expressed as %) are reported in panels (C), (F) and (I). Open bars represent results obtained from samples processed without an ultracentrifugation step while filled bars represent results obtained from samples treated with an ultracentrifugation step. The results from each experiment are expressed separately in each graphic. Labeling of two sets of data with different letters indicates that these two sets of data are statistically different ( P

    Article Snippet: 2.3.2 Ethidium bromide monoazide and propidium monoazide treatments EMA and PMA (Biotium, Hayward, CA) were reconstituted according to manufacturer's recommendation.

    Techniques: Next-Generation Sequencing, Labeling

    Cytokines production by reactivated splenocytes from mice treated with recombinant bacteria and challenged with TC-1 cells. Splenocytes were stimulated for 48 h with PMA ionomycin before measuring cytokines levels. Data represented mean ± SEM from 4 ( L. lactis wt) to 8 mice ( L. lactis IL-17). Data are analyzed with unpaired t -test followed by Mann–Withney post-test.

    Journal: Frontiers in Microbiology

    Article Title: Anti-tumoral Effects of Recombinant Lactococcus lactis Strain Secreting IL-17A Cytokine

    doi: 10.3389/fmicb.2018.03355

    Figure Lengend Snippet: Cytokines production by reactivated splenocytes from mice treated with recombinant bacteria and challenged with TC-1 cells. Splenocytes were stimulated for 48 h with PMA ionomycin before measuring cytokines levels. Data represented mean ± SEM from 4 ( L. lactis wt) to 8 mice ( L. lactis IL-17). Data are analyzed with unpaired t -test followed by Mann–Withney post-test.

    Article Snippet: For stimulation experiments, 1 × 106 cells per well were stimulated for 48 h (37°C, 10% CO2) in DMEM medium in P24 plates in presence of PMA (phorbol 12-myristate 13-acetate) ionomycin cocktail 1× (eBioscience).

    Techniques: Mouse Assay, Recombinant

    CD39+CD103+ T RM cells sorted from human endometrial tumors are transcriptionally active. ( A ) Overview of experimental set-up. CD39+CD103+ T RM cells and CD8+ TIL were sorted from human high-grade endometrial cancer digests ( n = 3). Subsequently, cells remained untreated or were treated for 4.5 h with actinomycin D, or 4 h of PMA/ionomyin or pre-incubation for 30 min with actinomycin D followed by 4 h PMA/ionomycin. ( B ) Concentration of cDNA (ng/μL/100 cells) of CD39+CD103+ T RM cells and CD8+ TIL per treatment group. The median concentration + 95% confidence interval is depicted. ( C ) Principal component analysis of mRNA sequencing data of all T RM samples. The first two principal components are depicted. Individual patient samples are identified by separate colors. ( D ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of untreated CD39+CD103+ T RM cells from three patients. The scale applies to all similar heatmaps of CD39+CD103+ T RM cells throughout the paper.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer

    doi: 10.3390/ijms21113770

    Figure Lengend Snippet: CD39+CD103+ T RM cells sorted from human endometrial tumors are transcriptionally active. ( A ) Overview of experimental set-up. CD39+CD103+ T RM cells and CD8+ TIL were sorted from human high-grade endometrial cancer digests ( n = 3). Subsequently, cells remained untreated or were treated for 4.5 h with actinomycin D, or 4 h of PMA/ionomyin or pre-incubation for 30 min with actinomycin D followed by 4 h PMA/ionomycin. ( B ) Concentration of cDNA (ng/μL/100 cells) of CD39+CD103+ T RM cells and CD8+ TIL per treatment group. The median concentration + 95% confidence interval is depicted. ( C ) Principal component analysis of mRNA sequencing data of all T RM samples. The first two principal components are depicted. Individual patient samples are identified by separate colors. ( D ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of untreated CD39+CD103+ T RM cells from three patients. The scale applies to all similar heatmaps of CD39+CD103+ T RM cells throughout the paper.

    Article Snippet: Half of each sample was incubated with PMA/ionomycin (eBioscience 1× Cell Stimulation Cocktail) for 4 h at room temperature, and all samples were incubated with a protein transport inhibitor to enable intracellular cytokine staining (1:1000, BD GolgiPlug™ Protein Transport Inhibitor, Thermo Fisher Scientific, BD555029, Waltham, MA, USA).

    Techniques: Incubation, Concentration Assay, Sequencing, Transformation Assay

    CD39+CD103+ T RM cells differentially upregulate genes associated with immunity, cell cycle, and the tumor microenvironment. ( A ) Volcano plot depicting differentially expressed genes (DEseq2) of CD39+CD103+ T RM cells treated with PMA/ionomycin for four hours versus untreated cells. Each dot represents a gene with a Benjamini–Hochberg adjusted p -value

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer

    doi: 10.3390/ijms21113770

    Figure Lengend Snippet: CD39+CD103+ T RM cells differentially upregulate genes associated with immunity, cell cycle, and the tumor microenvironment. ( A ) Volcano plot depicting differentially expressed genes (DEseq2) of CD39+CD103+ T RM cells treated with PMA/ionomycin for four hours versus untreated cells. Each dot represents a gene with a Benjamini–Hochberg adjusted p -value

    Article Snippet: Half of each sample was incubated with PMA/ionomycin (eBioscience 1× Cell Stimulation Cocktail) for 4 h at room temperature, and all samples were incubated with a protein transport inhibitor to enable intracellular cytokine staining (1:1000, BD GolgiPlug™ Protein Transport Inhibitor, Thermo Fisher Scientific, BD555029, Waltham, MA, USA).

    Techniques:

    CD39+CD103+ T RM and CD8+ TIL robustly express cytokine genes upon stimulation with PMA/ionomycin. ( A ) Overview of log2-normalized counts of CD39+CD103+ T RM (red) and CD8+ TIL (blue). Expression is depicted per treatment condition for GAPDH (household gene) and a series of cytotoxic T cell cytokines. Bars depict the median. ( B ) Overview of Ct-values determined using qPCR of CD39+CD103+ T RM (red) and CD8+ TIL (blue) for the same conditions as depicted in A. qPCR for IL13 was not determined (N.D.). Bars depict the median. ( C ) Correlation plots of Ct-values versus normalized counts of CD39+CD103+ T RM (red) and CD8+ TIL (blue). The dashed line represents a reference correlation of 1 (x = y).

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer

    doi: 10.3390/ijms21113770

    Figure Lengend Snippet: CD39+CD103+ T RM and CD8+ TIL robustly express cytokine genes upon stimulation with PMA/ionomycin. ( A ) Overview of log2-normalized counts of CD39+CD103+ T RM (red) and CD8+ TIL (blue). Expression is depicted per treatment condition for GAPDH (household gene) and a series of cytotoxic T cell cytokines. Bars depict the median. ( B ) Overview of Ct-values determined using qPCR of CD39+CD103+ T RM (red) and CD8+ TIL (blue) for the same conditions as depicted in A. qPCR for IL13 was not determined (N.D.). Bars depict the median. ( C ) Correlation plots of Ct-values versus normalized counts of CD39+CD103+ T RM (red) and CD8+ TIL (blue). The dashed line represents a reference correlation of 1 (x = y).

    Article Snippet: Half of each sample was incubated with PMA/ionomycin (eBioscience 1× Cell Stimulation Cocktail) for 4 h at room temperature, and all samples were incubated with a protein transport inhibitor to enable intracellular cytokine staining (1:1000, BD GolgiPlug™ Protein Transport Inhibitor, Thermo Fisher Scientific, BD555029, Waltham, MA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Activated CD8+ T cells are polyfunctional T cells that are responsive to (re)activation. ( A ) t-stochastic neighbor embedding plots of flow cytometry data of three endometrial tumor digests stained for CD3, CD8, IFN-γ, TNF-α, IL-2, and Zombie Aqua (live/dead stain). ( B ) Exemplary flow cytometry image of CD8+ T cells stained for CD3, CD8, IFN-γ, TNF-α, and IL-2. ( C ) Endometrial digests stained as described in ( B ) ( n = 6). Percentages of untreated cells (blue) or cells activated by 4-h incubation with PMA/ionomycin (red) that produce no, one, two, or three of the depicted cytokines. Bars depict the median. ( D ) Endometrial cancer digests ( n = 7) stained for CD3, CD8, IFN-γ, TNF-α, IL-2, and Zombie Aqua; 4 of these digests were additionally stained for IL-13 and IL-21, and 3 digests for IL-10 and GM-CSF. Percentages of untreated CD8+ TIL and CD8+ TIL activated by 4-h incubation with PMA/ionomycin positive for either of the depicted cytokines. Bars depict the median. ( E ) Exemplary flow cytometry images of CD8+ TIL stimulated for 4 h with PMA/ionomycin as depicted in D. ( F ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of PMA/ionomycin- and PMA/ionomycin + actinomycin D-treated CD39+CD103+ T RM cells.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer

    doi: 10.3390/ijms21113770

    Figure Lengend Snippet: Activated CD8+ T cells are polyfunctional T cells that are responsive to (re)activation. ( A ) t-stochastic neighbor embedding plots of flow cytometry data of three endometrial tumor digests stained for CD3, CD8, IFN-γ, TNF-α, IL-2, and Zombie Aqua (live/dead stain). ( B ) Exemplary flow cytometry image of CD8+ T cells stained for CD3, CD8, IFN-γ, TNF-α, and IL-2. ( C ) Endometrial digests stained as described in ( B ) ( n = 6). Percentages of untreated cells (blue) or cells activated by 4-h incubation with PMA/ionomycin (red) that produce no, one, two, or three of the depicted cytokines. Bars depict the median. ( D ) Endometrial cancer digests ( n = 7) stained for CD3, CD8, IFN-γ, TNF-α, IL-2, and Zombie Aqua; 4 of these digests were additionally stained for IL-13 and IL-21, and 3 digests for IL-10 and GM-CSF. Percentages of untreated CD8+ TIL and CD8+ TIL activated by 4-h incubation with PMA/ionomycin positive for either of the depicted cytokines. Bars depict the median. ( E ) Exemplary flow cytometry images of CD8+ TIL stimulated for 4 h with PMA/ionomycin as depicted in D. ( F ) Heatmap of a customized set of T cell markers of library size-normalized, log2-transformed counts of PMA/ionomycin- and PMA/ionomycin + actinomycin D-treated CD39+CD103+ T RM cells.

    Article Snippet: Half of each sample was incubated with PMA/ionomycin (eBioscience 1× Cell Stimulation Cocktail) for 4 h at room temperature, and all samples were incubated with a protein transport inhibitor to enable intracellular cytokine staining (1:1000, BD GolgiPlug™ Protein Transport Inhibitor, Thermo Fisher Scientific, BD555029, Waltham, MA, USA).

    Techniques: Flow Cytometry, Staining, Incubation, Transformation Assay

    SynGAP Regulates ERK Signaling to Control Excitatory Synaptic Strength. (A) Example images and the normalized mean (+SEM) intensity of AHA signals are presented from experiments using FUNCAT to measure protein synthesis rates. SynGAPsiRNA-mediated increase in AHA-incorporation rates was suppressed by treatment with the ERK blocker U0126 (25 µM, 6 hr). Scale bar = 5 µm. (B) Knockdown of SynGAP also increased levels of phosphorylated P70S6K, and this increase was suppressed by U0126. (C) U0126 rescued the increase in mEPSC amplitudes in neurons transfected with SynGAPsiRNA to control levels. The PMA-induced increase in mEPSC amplitudes in control neurons was occluded in neurons expressing SynGAPsiRNA. The increase in mEPSC amplitudes in cells expressing SynGAPsiRNA was not affected by the PKA blocker PKI 14–22 (1 µM, 6 hr). Note: the GFP control data in (C), shown for comparison, are also presented in Figure 2C .

    Journal: PLoS ONE

    Article Title: SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks

    doi: 10.1371/journal.pone.0083941

    Figure Lengend Snippet: SynGAP Regulates ERK Signaling to Control Excitatory Synaptic Strength. (A) Example images and the normalized mean (+SEM) intensity of AHA signals are presented from experiments using FUNCAT to measure protein synthesis rates. SynGAPsiRNA-mediated increase in AHA-incorporation rates was suppressed by treatment with the ERK blocker U0126 (25 µM, 6 hr). Scale bar = 5 µm. (B) Knockdown of SynGAP also increased levels of phosphorylated P70S6K, and this increase was suppressed by U0126. (C) U0126 rescued the increase in mEPSC amplitudes in neurons transfected with SynGAPsiRNA to control levels. The PMA-induced increase in mEPSC amplitudes in control neurons was occluded in neurons expressing SynGAPsiRNA. The increase in mEPSC amplitudes in cells expressing SynGAPsiRNA was not affected by the PKA blocker PKI 14–22 (1 µM, 6 hr). Note: the GFP control data in (C), shown for comparison, are also presented in Figure 2C .

    Article Snippet: U0126, PMA and an inactive version of PMA, P-2170 were from LC Laboratories (Woburn, BA).

    Techniques: Transfection, Expressing

    ERK Positively Regulates Synaptic AMPAR Content by Increasing Protein Synthesis via mTOR. (A) Representative mEPSC traces and combined data from cells in the presence or absence of the ERK blocker U0126 (25 µM, 6 hr) or ERK activator phorbol 12-myristate 13-acetate (PMA; 1 µg/ml, 1 hr). ERK blockade suppressed mEPSC amplitudes, whereas ERK activation increased mEPSC amplitudes in a manner that could be suppressed by U0126 or the mTOR antagonist KU-0063794 (KU). For co-treatment experiments cells were preincubated with U0126 or KU-0063794 before PMA application. (B) Example images and quantification from FUNCAT experiments of cortical neurons treated with U0126, PMA, or an inactive PMA analog, P-2170. Scale bar = 5 µm. PMA-induced ERK activation resulted in a significant increase in synthesis of dendritic protein. (C) ERK regulates synaptic AMPAR content in an mTOR-dependent manner. Overexpression of mTOR produced a significant increase in mEPSC amplitudes, which was suppressed by rapamycin or U0126, while any further PMA-mediated increase in synaptic AMPAR content was occluded in neurons overexpressing mTOR. (D) mTOR is a downstream effector of ERK in controlling protein synthesis. mTOR overexpression produced a significant increase in rates of protein synthesis as evidenced by the increase in AHA-incorporation rates. This mTOR mediated increase in protein synthesis rates was suppressed by the ERK blocker U0126.

    Journal: PLoS ONE

    Article Title: SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks

    doi: 10.1371/journal.pone.0083941

    Figure Lengend Snippet: ERK Positively Regulates Synaptic AMPAR Content by Increasing Protein Synthesis via mTOR. (A) Representative mEPSC traces and combined data from cells in the presence or absence of the ERK blocker U0126 (25 µM, 6 hr) or ERK activator phorbol 12-myristate 13-acetate (PMA; 1 µg/ml, 1 hr). ERK blockade suppressed mEPSC amplitudes, whereas ERK activation increased mEPSC amplitudes in a manner that could be suppressed by U0126 or the mTOR antagonist KU-0063794 (KU). For co-treatment experiments cells were preincubated with U0126 or KU-0063794 before PMA application. (B) Example images and quantification from FUNCAT experiments of cortical neurons treated with U0126, PMA, or an inactive PMA analog, P-2170. Scale bar = 5 µm. PMA-induced ERK activation resulted in a significant increase in synthesis of dendritic protein. (C) ERK regulates synaptic AMPAR content in an mTOR-dependent manner. Overexpression of mTOR produced a significant increase in mEPSC amplitudes, which was suppressed by rapamycin or U0126, while any further PMA-mediated increase in synaptic AMPAR content was occluded in neurons overexpressing mTOR. (D) mTOR is a downstream effector of ERK in controlling protein synthesis. mTOR overexpression produced a significant increase in rates of protein synthesis as evidenced by the increase in AHA-incorporation rates. This mTOR mediated increase in protein synthesis rates was suppressed by the ERK blocker U0126.

    Article Snippet: U0126, PMA and an inactive version of PMA, P-2170 were from LC Laboratories (Woburn, BA).

    Techniques: Activation Assay, Over Expression, Produced

    Rheb is a Downstream Effector of ERK and SynGAP Signaling to Regulate Excitatory Synaptic Strength. (A) Representative images of neurons stained with anti-Rheb-GTP antibody or total Rheb antibody in the presence or absence of the ERK blocker U0126 (25 µM, 6 hr). U0126 significantly suppressed levels of active Rheb without changing total Rheb levels. Scale bar = 5 µm. (B) Example images and normalized AHA intensity values from experiments using FUNCAT to measure protein synthesis rates. Overexpression of Rheb in cortical neurons produced a significant increase in protein synthesis rates as evidenced by an increase in AHA-labeling rates. This increase in protein synthesis was suppressed by treatment with U0126 (25 µM, 6 hr). Scale bar = 5 µm. (C) Overexpression of Rheb increased mEPSC amplitudes and was recovered by the treatment with U0126 (25 µM, 6 hr) or rapamycin (1 µM, 6 hr). Treatment with PMA (1 µg/ml, 6 hr) did not cause a further increase in mEPSC amplitudes in Rheb overexpressing neurons. Co-expression of Rheb did not produce a further increase in AMPAR-mEPSCs in neurons expressing SynGAPsiRNA.

    Journal: PLoS ONE

    Article Title: SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks

    doi: 10.1371/journal.pone.0083941

    Figure Lengend Snippet: Rheb is a Downstream Effector of ERK and SynGAP Signaling to Regulate Excitatory Synaptic Strength. (A) Representative images of neurons stained with anti-Rheb-GTP antibody or total Rheb antibody in the presence or absence of the ERK blocker U0126 (25 µM, 6 hr). U0126 significantly suppressed levels of active Rheb without changing total Rheb levels. Scale bar = 5 µm. (B) Example images and normalized AHA intensity values from experiments using FUNCAT to measure protein synthesis rates. Overexpression of Rheb in cortical neurons produced a significant increase in protein synthesis rates as evidenced by an increase in AHA-labeling rates. This increase in protein synthesis was suppressed by treatment with U0126 (25 µM, 6 hr). Scale bar = 5 µm. (C) Overexpression of Rheb increased mEPSC amplitudes and was recovered by the treatment with U0126 (25 µM, 6 hr) or rapamycin (1 µM, 6 hr). Treatment with PMA (1 µg/ml, 6 hr) did not cause a further increase in mEPSC amplitudes in Rheb overexpressing neurons. Co-expression of Rheb did not produce a further increase in AMPAR-mEPSCs in neurons expressing SynGAPsiRNA.

    Article Snippet: U0126, PMA and an inactive version of PMA, P-2170 were from LC Laboratories (Woburn, BA).

    Techniques: Staining, Over Expression, Produced, Labeling, Expressing

    ERK and Rheb are Downstream of GluN2B Signaling. (A) GluN2B knockout neurons revealed a significant increase in protein synthesis rates as evidenced by measuring AHA incorporation rates. This increase was suppressed by treatment with U0126 (25 µM, 6 hr), scale bar = 5 µm. (B) U0126 also suppressed the increase in mEPSC amplitudes observed in non-treated in GluN2B knockout neurons. Activation of ERK activity using PMA did not cause any further increase in mEPSC amplitudes in GluN2B knockout neurons. (C) The Rheb overexpression-induced increase in mEPSC amplitudes observed in WT neurons was occluded in GluN2B knockout neurons. (D) Rheb-GTP levels, determined using anti-Rheb-GTP antibody, increased in GluN2B knockout neurons. The total levels of Rheb signal were reduced in the absence of GluN2B.

    Journal: PLoS ONE

    Article Title: SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks

    doi: 10.1371/journal.pone.0083941

    Figure Lengend Snippet: ERK and Rheb are Downstream of GluN2B Signaling. (A) GluN2B knockout neurons revealed a significant increase in protein synthesis rates as evidenced by measuring AHA incorporation rates. This increase was suppressed by treatment with U0126 (25 µM, 6 hr), scale bar = 5 µm. (B) U0126 also suppressed the increase in mEPSC amplitudes observed in non-treated in GluN2B knockout neurons. Activation of ERK activity using PMA did not cause any further increase in mEPSC amplitudes in GluN2B knockout neurons. (C) The Rheb overexpression-induced increase in mEPSC amplitudes observed in WT neurons was occluded in GluN2B knockout neurons. (D) Rheb-GTP levels, determined using anti-Rheb-GTP antibody, increased in GluN2B knockout neurons. The total levels of Rheb signal were reduced in the absence of GluN2B.

    Article Snippet: U0126, PMA and an inactive version of PMA, P-2170 were from LC Laboratories (Woburn, BA).

    Techniques: Knock-Out, Activation Assay, Activity Assay, Over Expression

    Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with PMA+ionomycin. In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with PMA+ionomycin. In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques:

    Percentage of cytokine-producing T cells after short-term stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of the frequencies of monofunctional and multi-functional CD4 + and CD8 + T lymphocytes for tumour necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ after stimulation with PMA+ionomycin among individuals younger and older than 50 years of age. (b) Comparative analysis of T lymphocyte functionality between age groups after stimulation with EBV lysate. The middle line and the vertical lines represent the median and ranges, respectively. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Percentage of cytokine-producing T cells after short-term stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of the frequencies of monofunctional and multi-functional CD4 + and CD8 + T lymphocytes for tumour necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ after stimulation with PMA+ionomycin among individuals younger and older than 50 years of age. (b) Comparative analysis of T lymphocyte functionality between age groups after stimulation with EBV lysate. The middle line and the vertical lines represent the median and ranges, respectively. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques: Functional Assay

    Frequency of tumour necrosis factor (TNF)-α + T cells after in-vitro stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Frequency of CD4 + and CD8 + T cells (TNF-α + ) after polyclonal stimulation with PMA+ionomycin in the peripheral blood (PB) of healthy subjects grouped according to age. (b) Relative frequencies of activated CD4 + and CD8 + T cells (TNF-α + ) in the absence and presence of EBV lysate in individuals from the two age groups. (c) Comparison of the specific T cell response after stimulus with EBV lysate among healthy individuals. In all figures, the median and range are shown. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Frequency of tumour necrosis factor (TNF)-α + T cells after in-vitro stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Frequency of CD4 + and CD8 + T cells (TNF-α + ) after polyclonal stimulation with PMA+ionomycin in the peripheral blood (PB) of healthy subjects grouped according to age. (b) Relative frequencies of activated CD4 + and CD8 + T cells (TNF-α + ) in the absence and presence of EBV lysate in individuals from the two age groups. (c) Comparison of the specific T cell response after stimulus with EBV lysate among healthy individuals. In all figures, the median and range are shown. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques: In Vitro

    Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), phorbol-12-myristate-13-acetate (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P

    Journal: Journal of Bone Metabolism

    Article Title: Bone Morphogenetic Protein-2 Desensitizes MC3T3-E1 Osteoblastic Cells to Estrogen Through Transcriptional Downregulation of Estrogen Receptor 1

    doi: 10.11005/jbm.2013.20.2.83

    Figure Lengend Snippet: Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D 3 (1,25-[OH] 2 D 3 ; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), phorbol-12-myristate-13-acetate (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. * P

    Article Snippet: Phorbol-12-myristate-13-acetate (PMA) and dibutyryl cyclic adenosine monophosphate (dbcAMP) were purchased from Wako (Osaka, Japan).

    Techniques: Activity Assay, Recombinant, Incubation, Luciferase, Transfection, Lysis

    Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with DMSO, 5 µM nutlin, PMA, or nutlin + PMA. Statistically significant changes by Student’s t test ( P

    Journal: mBio

    Article Title: Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and TP53-Independent Mechanism

    doi: 10.1128/mBio.02690-18

    Figure Lengend Snippet: Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells treated with DMSO, 5 µM nutlin, PMA, or nutlin + PMA. Statistically significant changes by Student’s t test ( P

    Article Snippet: For pretreatment with PMA (tlrl-pma; InvivoGen; 1-mg/ml stock in DMSO), cells were treated with 0 ng/ml (DMSO) or 25 ng/ml PMA for 3 h, followed by treatment with 0 µM, 0.5 µM, and 2.5 µM palbociclib.

    Techniques: Expressing, Quantitative RT-PCR

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-07556-3

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

    Article Snippet: Phorbol-12-myristate-13-acetate (PMA), 4α-phorbol 12,13-didecanoate (4αPDD) and ionomycin were purchased from Merck Millipore.

    Techniques: Transfection, SDS Page

    Ser1134 and Ser1161 are the PMA- and EGF-induced SOS1 phosphorylation sites that conform to the minimal RSK consensus motif. ( A ) HEK 293 cells were treated as described in and whole cell extracts as well as ten percent of each immune complex

    Journal: The Biochemical journal

    Article Title: RSK phosphorylates SOS1 creating 14-3-3 docking sites and negatively regulating MAPK activation

    doi: 10.1042/BJ20120938

    Figure Lengend Snippet: Ser1134 and Ser1161 are the PMA- and EGF-induced SOS1 phosphorylation sites that conform to the minimal RSK consensus motif. ( A ) HEK 293 cells were treated as described in and whole cell extracts as well as ten percent of each immune complex

    Article Snippet: Pharmacological inhibitors and stimulants were from the following sources (with final concentrations indicated): BI-D1870 (10 μM; Biomol, Plymouth Meeting, PA), SL0101 (50 μM; Toronto Research Chemicals), UO126 (20 μM; Biomol), PD184352 (10 μM; Calbiochem, San Diego, CA), PMA (25 ng/ml; Biomol and Calbiochem) EGF (25 ng/ml; Invitrogen, Carlsbad, CA, and Cell signaling Technology, Beverly, MA).

    Techniques:

    Identification of one or more potential RSK phosphorylation sites in SOS1 using an anti-RXXpS-specific motif antibody. ( A ) PMA and EGF induce phosphorylation of an RXXS motif in SOS1. HEK 293 cells transfected with HA-SOS1 were starved of serum and left

    Journal: The Biochemical journal

    Article Title: RSK phosphorylates SOS1 creating 14-3-3 docking sites and negatively regulating MAPK activation

    doi: 10.1042/BJ20120938

    Figure Lengend Snippet: Identification of one or more potential RSK phosphorylation sites in SOS1 using an anti-RXXpS-specific motif antibody. ( A ) PMA and EGF induce phosphorylation of an RXXS motif in SOS1. HEK 293 cells transfected with HA-SOS1 were starved of serum and left

    Article Snippet: Pharmacological inhibitors and stimulants were from the following sources (with final concentrations indicated): BI-D1870 (10 μM; Biomol, Plymouth Meeting, PA), SL0101 (50 μM; Toronto Research Chemicals), UO126 (20 μM; Biomol), PD184352 (10 μM; Calbiochem, San Diego, CA), PMA (25 ng/ml; Biomol and Calbiochem) EGF (25 ng/ml; Invitrogen, Carlsbad, CA, and Cell signaling Technology, Beverly, MA).

    Techniques: Transfection