Structured Review

Becton Dickinson phospho erk
SKAP-55 deficient T-cells show enhanced anti-CD3 induced <t>ERK</t> activation. Panel A: <t>FACS</t> profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .
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1) Product Images from "Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells"

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001718

SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .
Figure Legend Snippet: SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .

Techniques Used: Activation Assay, FACS, Mouse Assay, Staining, Labeling, Binding Assay

2) Product Images from "Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils"

Article Title: Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124082

Mechanisms involved in RSV Fusion protein-induced NET formation in human neutrophils. (I) RSV F protein binds to and activates TLR-4, expressed by neutrophils, stimulating ROS production via NADPH Oxidase, which is essential for NET formation. (II) F protein is also able to activate ERK and p38 MAPK to induce NET release. RSV F protein stimulates the production of NETs decorated with the granular proteins NE and MPO.
Figure Legend Snippet: Mechanisms involved in RSV Fusion protein-induced NET formation in human neutrophils. (I) RSV F protein binds to and activates TLR-4, expressed by neutrophils, stimulating ROS production via NADPH Oxidase, which is essential for NET formation. (II) F protein is also able to activate ERK and p38 MAPK to induce NET release. RSV F protein stimulates the production of NETs decorated with the granular proteins NE and MPO.

Techniques Used:

F protein activates ERK and p38 MAPK to induce NET formation. (A,B) Neutrophils (2 x 10 6 /mL) were pretreated with PD98059 (30 μM) or SB203580 (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO 2 . NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p
Figure Legend Snippet: F protein activates ERK and p38 MAPK to induce NET formation. (A,B) Neutrophils (2 x 10 6 /mL) were pretreated with PD98059 (30 μM) or SB203580 (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO 2 . NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p

Techniques Used:

3) Product Images from "Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes"

Article Title: Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes

Journal: Clinical Science (London, England : 1979)

doi: 10.1042/CS20100084

Effect of signalling pathway inhibitors on hypoxia-induced myocardin expression and ERK phosphorylation (A and B) ERK pathway mediates hypoxia-induced myocardin expression in neonatal cardiomyocytes. Neonatal cardiomyocytes were pre-treated with an ERK pathway inhibitor (PD98059), a JNK inhibitor (SP600125), a p38 MAPK inhibitor (SB203580), a PI3K/Akt inhibitor (wortmannin) or ERK siRNA, followed by hypoxia for 4 h. Neonatal cardiomyocytes were harvested and cell lysates were analysed by Western blotting using an anti-myocardin antibody. Result are normalized to actin levels. * P
Figure Legend Snippet: Effect of signalling pathway inhibitors on hypoxia-induced myocardin expression and ERK phosphorylation (A and B) ERK pathway mediates hypoxia-induced myocardin expression in neonatal cardiomyocytes. Neonatal cardiomyocytes were pre-treated with an ERK pathway inhibitor (PD98059), a JNK inhibitor (SP600125), a p38 MAPK inhibitor (SB203580), a PI3K/Akt inhibitor (wortmannin) or ERK siRNA, followed by hypoxia for 4 h. Neonatal cardiomyocytes were harvested and cell lysates were analysed by Western blotting using an anti-myocardin antibody. Result are normalized to actin levels. * P

Techniques Used: Expressing, Western Blot

4) Product Images from "A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells"

Article Title: A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2013/658370

The propolis derivative, GS-002, induced ATF-3 expression that was mediated by MAPK pathways. Hep3B cells were treated with (a) 20 μ g/mL GS-002 for the indicated time periods, or (b) with various concentrations of GS-002 for 1 h. Total cell lysates were used to detect protein expressions of p38, phosphor-p38 (p-p38), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), extracellular signal-regulated kinase (ERK), and phospho-ERK (p-ERK) by Western blotting. Ten micrograms of 15-deoxy-Δ12,14-prostaglandin J 2 (15d-PGJ 2 ) was used as a positive control. (c) Hep3B cells were pretreated with 5 and 10 μ M of the p38 inhibitor, SB203580, the ERK inhibitor, PD98059, or the JNK inhibitor, SP600125, for 90 min, then treated with GS-002 (20 μ g/mL) for another 12 h, and the ATF-3 protein level was detected by Western blotting.
Figure Legend Snippet: The propolis derivative, GS-002, induced ATF-3 expression that was mediated by MAPK pathways. Hep3B cells were treated with (a) 20 μ g/mL GS-002 for the indicated time periods, or (b) with various concentrations of GS-002 for 1 h. Total cell lysates were used to detect protein expressions of p38, phosphor-p38 (p-p38), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), extracellular signal-regulated kinase (ERK), and phospho-ERK (p-ERK) by Western blotting. Ten micrograms of 15-deoxy-Δ12,14-prostaglandin J 2 (15d-PGJ 2 ) was used as a positive control. (c) Hep3B cells were pretreated with 5 and 10 μ M of the p38 inhibitor, SB203580, the ERK inhibitor, PD98059, or the JNK inhibitor, SP600125, for 90 min, then treated with GS-002 (20 μ g/mL) for another 12 h, and the ATF-3 protein level was detected by Western blotting.

Techniques Used: Expressing, Western Blot, Positive Control

5) Product Images from "Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex"

Article Title: Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.3.907-920.2005

Phospho-Ser 676 antibodies reveal NFATc4 phosphorylation. (A) Phosphorylation of NFATc4 Ser 676 by ERK in vitro. Recombinant NFATc4 (GST-NFATc4 531-749) was phosphorylated by ERK in vitro by using immune complex kinase assays. Phosphorylation of NFATc4 Ser 676 was detected by immunoblot analysis (IB) with anti-phosphoNFATc4 (p-Ser676) and GST antibodies. ns, nonspecific cross-reaction. (B) Phosphorylation of Ser 676 by ERK is susceptible to U0126. HA-tagged NFATc4 REL domain (NFATc4 344-749) was transfected into COS cells, and extracts were prepared from PMA-treated (+; 100 nM) or untreated (−) cells for immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. The expression of activated (p-ERK) and total ERK is indicated. The effect of replacement of Ser 676 with Ala and pretreatment with MEK inhibitor U0126 were also examined. (C) Phosphorylation of Ser 676 by RSK2. NFATc4 was coexpressed with constitutive-active RSK2 or constitutive-active MEK1 in COS cells. Cell extracts were examined by immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. Expression of RSK2 and MEK1 was indicated. (D) Phosphorylation of Ser 676 of endogenous NFATc4. Endogenous NFATc4 were immunoprecipitated from untreated (−) or PMA-treated (+) Jurkat cell extracts. DNA affinity isolation was also performed by using the PPARγ2 proximal NFAT element to precipitate endogenous NFAT. Phosphorylation of Ser 676 of NFATc4 (p-Ser676) in the immunoprecipitates and DNA precipitates was detected by immunoblot analysis. The effect of pretreatment with MEK inhibitor U0126 was also examined. Activation (p-RSK) and expression of RSK in the cell lysate was also shown.
Figure Legend Snippet: Phospho-Ser 676 antibodies reveal NFATc4 phosphorylation. (A) Phosphorylation of NFATc4 Ser 676 by ERK in vitro. Recombinant NFATc4 (GST-NFATc4 531-749) was phosphorylated by ERK in vitro by using immune complex kinase assays. Phosphorylation of NFATc4 Ser 676 was detected by immunoblot analysis (IB) with anti-phosphoNFATc4 (p-Ser676) and GST antibodies. ns, nonspecific cross-reaction. (B) Phosphorylation of Ser 676 by ERK is susceptible to U0126. HA-tagged NFATc4 REL domain (NFATc4 344-749) was transfected into COS cells, and extracts were prepared from PMA-treated (+; 100 nM) or untreated (−) cells for immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. The expression of activated (p-ERK) and total ERK is indicated. The effect of replacement of Ser 676 with Ala and pretreatment with MEK inhibitor U0126 were also examined. (C) Phosphorylation of Ser 676 by RSK2. NFATc4 was coexpressed with constitutive-active RSK2 or constitutive-active MEK1 in COS cells. Cell extracts were examined by immunoblot analysis with anti-phosphoNFATc4 (p-Ser676) and HA antibodies. Expression of RSK2 and MEK1 was indicated. (D) Phosphorylation of Ser 676 of endogenous NFATc4. Endogenous NFATc4 were immunoprecipitated from untreated (−) or PMA-treated (+) Jurkat cell extracts. DNA affinity isolation was also performed by using the PPARγ2 proximal NFAT element to precipitate endogenous NFAT. Phosphorylation of Ser 676 of NFATc4 (p-Ser676) in the immunoprecipitates and DNA precipitates was detected by immunoblot analysis. The effect of pretreatment with MEK inhibitor U0126 was also examined. Activation (p-RSK) and expression of RSK in the cell lysate was also shown.

Techniques Used: In Vitro, Recombinant, Immune Complex Kinase Assay, Transfection, Expressing, Immunoprecipitation, Isolation, Activation Assay

RSK and ERK phosphorylates Ser 676 of the NFATc4 REL homology domain. (A) Phosphorylation of Ser 676 of NFATc4 by RSK2. Recombinant NFATc4 REL homology domain (GST-NFATc4 531-749) is illustrated schematically. Replacement of Ser 676 with Ala is also indicated. Epitope-tagged RSK2 was transfected into COS cells, and extracts were prepared from PMA-treated cells (+; 100 nM) or untreated cells (−) for immune complex kinase assays. Phosphorylation of NFATc4 by RSK2 was quantitated by phosphorimager analysis. The effect of mutation of Ser 676 (NFATc4 531-749 S 676 A) on NFATc4 phosphorylation was also examined. (B) Phosphorylation of Ser 676 of NFATc4 by ERK. Epitope-tagged ERK, which was activated (+) or not activated (−) by coexpression with constitutive-active MEK1, was immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of GST-NFATc4 531-749 by ERK was examined by immune complex kinase assays and quantitated by phosphorimager analysis. The effect of mutation at Ser 676 (NFATc4 531-749 S 676 A) on NFATc4 phosphorylation was also examined. (C) Epitope-tagged JNK, ERK, and p38 MAPK, which were activatedor not by coexpression with MLK3, constitutive-active MEK1, and constitutive-active MKK6, respectively, were immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of NFATc4 DNA-binding domain was examined by autoradiography.
Figure Legend Snippet: RSK and ERK phosphorylates Ser 676 of the NFATc4 REL homology domain. (A) Phosphorylation of Ser 676 of NFATc4 by RSK2. Recombinant NFATc4 REL homology domain (GST-NFATc4 531-749) is illustrated schematically. Replacement of Ser 676 with Ala is also indicated. Epitope-tagged RSK2 was transfected into COS cells, and extracts were prepared from PMA-treated cells (+; 100 nM) or untreated cells (−) for immune complex kinase assays. Phosphorylation of NFATc4 by RSK2 was quantitated by phosphorimager analysis. The effect of mutation of Ser 676 (NFATc4 531-749 S 676 A) on NFATc4 phosphorylation was also examined. (B) Phosphorylation of Ser 676 of NFATc4 by ERK. Epitope-tagged ERK, which was activated (+) or not activated (−) by coexpression with constitutive-active MEK1, was immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of GST-NFATc4 531-749 by ERK was examined by immune complex kinase assays and quantitated by phosphorimager analysis. The effect of mutation at Ser 676 (NFATc4 531-749 S 676 A) on NFATc4 phosphorylation was also examined. (C) Epitope-tagged JNK, ERK, and p38 MAPK, which were activatedor not by coexpression with MLK3, constitutive-active MEK1, and constitutive-active MKK6, respectively, were immunoprecipitated and incubated with recombinant GST-NFATc4 531-749 proteins. Phosphorylation of NFATc4 DNA-binding domain was examined by autoradiography.

Techniques Used: Recombinant, Transfection, Immune Complex Kinase Assay, Mutagenesis, Immunoprecipitation, Incubation, Binding Assay, Autoradiography

ERK binds to NFATc4. (A) Endogenous NFATc4 interacts with activated ERK. The ERK docking FxF motif on NFAT REL domains is illustrated schematically. The position of the β-strands (β f and β g ), is also shown. Ser 676 of NFATc4 is also highlighted. Endogenous NFATc4 were immunoprecipitated (IP) from untreated or ionomycin (Ion; 2 μM) and phorbol ester (PMA; 100 nM) treated (Ion+PMA) mouse embryonic fibroblast extracts. Endogenous ERK in the immunoprecipitates was detected by immunoblot (IB) analysis. Activation (p-ERK) and expression of ERK in the cell lysate was also shown. (B) Identification of ERK binding site on NFATc4. Various COOH-terminal truncated NFATc4 proteins (residues 1 to 902, 1 to 853, 1 to 581, 1 to 450, 1 to 365, 1 to 308, and 1 to 260) were coexpressed with epitope-tagged ERK in COS cells. NFATc4 proteins in the extracts and immunoprecipitated (IP) with ERK were detected by immunoblot (IB) analysis. A schematic illustration of the NFATc4 protein and the ERK binding site are also shown. (C) Extracts prepared from cells transfected with epitope-tagged NFATc4 344-749 were incubated with recombinant GST-ERK. NFATc4 in the extracts and bound to the immobilized GST-ERK were detected by immunoblot analysis. Effect of deletion (NFATc4 344-581) and mutation of the ERK FxF docking site (NFATc4 344-749 FF 681,683 GG) on NFATc4 interaction was also examined. (D) Recombinant GST and GST-NFATc4 531-749 proteins were incubated with extracts prepared from cells transfected with epitope-tagged ERK. ERK bound to the immobilized GST proteins was detected by immunoblot analysis. The effect of mutation of the ERK FxF docking site (NFATc4 531-749 FF 681,683 GG) on NFATc4 interaction was also examined.
Figure Legend Snippet: ERK binds to NFATc4. (A) Endogenous NFATc4 interacts with activated ERK. The ERK docking FxF motif on NFAT REL domains is illustrated schematically. The position of the β-strands (β f and β g ), is also shown. Ser 676 of NFATc4 is also highlighted. Endogenous NFATc4 were immunoprecipitated (IP) from untreated or ionomycin (Ion; 2 μM) and phorbol ester (PMA; 100 nM) treated (Ion+PMA) mouse embryonic fibroblast extracts. Endogenous ERK in the immunoprecipitates was detected by immunoblot (IB) analysis. Activation (p-ERK) and expression of ERK in the cell lysate was also shown. (B) Identification of ERK binding site on NFATc4. Various COOH-terminal truncated NFATc4 proteins (residues 1 to 902, 1 to 853, 1 to 581, 1 to 450, 1 to 365, 1 to 308, and 1 to 260) were coexpressed with epitope-tagged ERK in COS cells. NFATc4 proteins in the extracts and immunoprecipitated (IP) with ERK were detected by immunoblot (IB) analysis. A schematic illustration of the NFATc4 protein and the ERK binding site are also shown. (C) Extracts prepared from cells transfected with epitope-tagged NFATc4 344-749 were incubated with recombinant GST-ERK. NFATc4 in the extracts and bound to the immobilized GST-ERK were detected by immunoblot analysis. Effect of deletion (NFATc4 344-581) and mutation of the ERK FxF docking site (NFATc4 344-749 FF 681,683 GG) on NFATc4 interaction was also examined. (D) Recombinant GST and GST-NFATc4 531-749 proteins were incubated with extracts prepared from cells transfected with epitope-tagged ERK. ERK bound to the immobilized GST proteins was detected by immunoblot analysis. The effect of mutation of the ERK FxF docking site (NFATc4 531-749 FF 681,683 GG) on NFATc4 interaction was also examined.

Techniques Used: Immunoprecipitation, Activation Assay, Expressing, Binding Assay, Transfection, Incubation, Recombinant, Mutagenesis

Activation of RSK and ERK potentiates NFAT-mediated gene transcription. (A) MEK inhibitor U0126 reduces NFAT-mediated transcription. Cells transfected with NFAT-luciferase reporter were pretreated or not with MEK inhibitor U0126 for 2 h. The cells were then stimulated without (untreated) and with serum (20%) plus ionomycin (Ion; 2 μM) for 16 h before harvest. Transfection efficiency was monitored by measurement of β-galactosidase activity. (B) Expression constructs for full-length, wild-type NFATc4, constitutive nuclear NFATc4 (NFATc4 SS 168,170 AA), and constitutive nuclear, Ser 676 phosphorylation-defective NFATc4 (NFATc4 SS 168,170 AA+S 676 A) were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and β-galactosidase activities were measured. (C) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749), ERK binding-defective NFATc4 344-749 FF 681,683 GG, and Ser 676 phosphorylation-defective NFATc4 344-749 S 676 A were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. Luciferase and β-galactosidase activities were measured. (D) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749) and cooperation-defective NFATc4 (NFATc4 344-749 RN 474,475 AA, T 541 G) were coexpressed with C/EBPβ and, in the presence or absence of constitutive-active MEK1. Compound mutation to abolish Ser 676 phosphorylation and NFAT cooperative interaction (NFATc4 344-749 RN 474,475 AA, T 541 G+S 676 A) was also examined. Cells were harvested 36 h after transfection. NFAT-mediated luciferase activity and control of β-galactosidase activity were measured. (E) Expression constructs for full-length, constitutive nuclear NFATc4 (NFATc4 SS 168,170 AA), and constitutive nuclear, Ser 676 phosphorylation-defective NFATc4 (NFATc4 SS 168,170 AA+S 676 A) were coexpressed, in the presence or absence of wild-type RSK2, constitutive-active RSK2 (active RSK2) or dead RSK2, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and β-galactosidase activities were measured.
Figure Legend Snippet: Activation of RSK and ERK potentiates NFAT-mediated gene transcription. (A) MEK inhibitor U0126 reduces NFAT-mediated transcription. Cells transfected with NFAT-luciferase reporter were pretreated or not with MEK inhibitor U0126 for 2 h. The cells were then stimulated without (untreated) and with serum (20%) plus ionomycin (Ion; 2 μM) for 16 h before harvest. Transfection efficiency was monitored by measurement of β-galactosidase activity. (B) Expression constructs for full-length, wild-type NFATc4, constitutive nuclear NFATc4 (NFATc4 SS 168,170 AA), and constitutive nuclear, Ser 676 phosphorylation-defective NFATc4 (NFATc4 SS 168,170 AA+S 676 A) were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and β-galactosidase activities were measured. (C) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749), ERK binding-defective NFATc4 344-749 FF 681,683 GG, and Ser 676 phosphorylation-defective NFATc4 344-749 S 676 A were coexpressed, in the presence or absence of constitutive-active MEK1, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. Luciferase and β-galactosidase activities were measured. (D) Expression constructs for NFATc4 REL DNA-binding domain (NFATc4 344-749) and cooperation-defective NFATc4 (NFATc4 344-749 RN 474,475 AA, T 541 G) were coexpressed with C/EBPβ and, in the presence or absence of constitutive-active MEK1. Compound mutation to abolish Ser 676 phosphorylation and NFAT cooperative interaction (NFATc4 344-749 RN 474,475 AA, T 541 G+S 676 A) was also examined. Cells were harvested 36 h after transfection. NFAT-mediated luciferase activity and control of β-galactosidase activity were measured. (E) Expression constructs for full-length, constitutive nuclear NFATc4 (NFATc4 SS 168,170 AA), and constitutive nuclear, Ser 676 phosphorylation-defective NFATc4 (NFATc4 SS 168,170 AA+S 676 A) were coexpressed, in the presence or absence of wild-type RSK2, constitutive-active RSK2 (active RSK2) or dead RSK2, with the NFAT-luciferase reporter plasmid. Cells were harvested 36 h after transfection. The luciferase and β-galactosidase activities were measured.

Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay, Expressing, Construct, Plasmid Preparation, Binding Assay, Mutagenesis

6) Product Images from "The FBW7-MCL-1 axis is key in M1 and M2 macrophage-related colon cancer cell progression: validating the immunotherapeutic value of targeting PI3Kγ"

Article Title: The FBW7-MCL-1 axis is key in M1 and M2 macrophage-related colon cancer cell progression: validating the immunotherapeutic value of targeting PI3Kγ

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-020-0436-7

Ex vivo analysis of the role of macrophage CM in colon cancer patient-derived cells (PDCs). a Estimation of PDC viability after coculture with macrophage CM for 24 h using the WST-1 assay. Error bars are derived from three independent experiments. b Expression levels of apoptosis and EMT markers in macrophage CM-treated PDCs were measured by western blotting. c Wound-healing ability was evaluated by creating wounds on a confluent monolayer of PDCs using 1-Dish 35-mm-high culture inserts. d Activation of AKT and ERK and expression levels of MCL-1 and FBW7 assessed by western blotting. Actin was used as a loading control. The results are presented as the mean ± SE. ** P
Figure Legend Snippet: Ex vivo analysis of the role of macrophage CM in colon cancer patient-derived cells (PDCs). a Estimation of PDC viability after coculture with macrophage CM for 24 h using the WST-1 assay. Error bars are derived from three independent experiments. b Expression levels of apoptosis and EMT markers in macrophage CM-treated PDCs were measured by western blotting. c Wound-healing ability was evaluated by creating wounds on a confluent monolayer of PDCs using 1-Dish 35-mm-high culture inserts. d Activation of AKT and ERK and expression levels of MCL-1 and FBW7 assessed by western blotting. Actin was used as a loading control. The results are presented as the mean ± SE. ** P

Techniques Used: Ex Vivo, Derivative Assay, WST-1 Assay, Expressing, Western Blot, Activation Assay

Inhibition of PI3Kγ attenuates tumor growth in xenograft models associated with infiltrated macrophages. a Experimental procedure illustrating the TG100-115 treatment regimen in BALB/c mice. b CT26 cells (5 × 10 5 cells/mouse) were subcutaneously injected into the flanks of 6-week-old mice. Mean tumor volume of subcutaneously implanted vehicle- or TG100-115-treated mice ( n = 5) and representative images of subcutaneous tumors at day 16 after treatment with vehicle or TG100-115 (box) are shown. c FACS analysis and quantification of CD11b + F4/80 + (TAM) cell populations in CT26 tumors at day 14 posttreatment ( n = 5) and expression levels of MHCII (M1) and CD206 (M2) in CD11b + F4/80 + cell populations. d Graph showing the percentage of each population (M1, M2) in the vehicle-treated group in comparison with the TG100-115-treated group. e mRNA expression levels of genes involved in the proinflammatory response ( il-1β, cxcl10 ) and antiinflammatory response ( il-10 and tgf-β ) were evaluated by real-time PCR in vehicle and TG100-155-treated groups. f Representative western blot analysis showing survival/EMT-related protein expression as well as ERK/AKT-FBW7-MCL-1 signal axis regulation in vehicle- and TG100-155-treated mouse tumor tissues.
Figure Legend Snippet: Inhibition of PI3Kγ attenuates tumor growth in xenograft models associated with infiltrated macrophages. a Experimental procedure illustrating the TG100-115 treatment regimen in BALB/c mice. b CT26 cells (5 × 10 5 cells/mouse) were subcutaneously injected into the flanks of 6-week-old mice. Mean tumor volume of subcutaneously implanted vehicle- or TG100-115-treated mice ( n = 5) and representative images of subcutaneous tumors at day 16 after treatment with vehicle or TG100-115 (box) are shown. c FACS analysis and quantification of CD11b + F4/80 + (TAM) cell populations in CT26 tumors at day 14 posttreatment ( n = 5) and expression levels of MHCII (M1) and CD206 (M2) in CD11b + F4/80 + cell populations. d Graph showing the percentage of each population (M1, M2) in the vehicle-treated group in comparison with the TG100-115-treated group. e mRNA expression levels of genes involved in the proinflammatory response ( il-1β, cxcl10 ) and antiinflammatory response ( il-10 and tgf-β ) were evaluated by real-time PCR in vehicle and TG100-155-treated groups. f Representative western blot analysis showing survival/EMT-related protein expression as well as ERK/AKT-FBW7-MCL-1 signal axis regulation in vehicle- and TG100-155-treated mouse tumor tissues.

Techniques Used: Inhibition, Mouse Assay, Injection, FACS, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Opposite effects of M1 macrophage CM and M2 macrophage CM on FBW7-mediated MCL-1 degradation of colon cancer cells. Expression levels of AKT, ERK, MCL-1, and FBW7 in a macrophage CM-treated HCT116 and HT29 cells and in b mouse subcutaneous tissues transplanted with long-term coculture and differentiated M0, M1, and M2 macrophages. c mRNA levels of MCL-1 and FBW7 were evaluated by RT-PCR after macrophage CM treatment for 24 h. d HT29 cells were exposed to MG132 for 1 h and incubated with macrophage CM for 24 h. e HCT116 cells were transfected with either empty vector or MCL-1 expression vector for 24 h, followed by incubation with macrophage CM for another 24 h. Actin was used as a loading control.
Figure Legend Snippet: Opposite effects of M1 macrophage CM and M2 macrophage CM on FBW7-mediated MCL-1 degradation of colon cancer cells. Expression levels of AKT, ERK, MCL-1, and FBW7 in a macrophage CM-treated HCT116 and HT29 cells and in b mouse subcutaneous tissues transplanted with long-term coculture and differentiated M0, M1, and M2 macrophages. c mRNA levels of MCL-1 and FBW7 were evaluated by RT-PCR after macrophage CM treatment for 24 h. d HT29 cells were exposed to MG132 for 1 h and incubated with macrophage CM for 24 h. e HCT116 cells were transfected with either empty vector or MCL-1 expression vector for 24 h, followed by incubation with macrophage CM for another 24 h. Actin was used as a loading control.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Transfection, Plasmid Preparation

7) Product Images from "A role for gut-associated lymphoid tissue in shaping the human B cell repertoire"

Article Title: A role for gut-associated lymphoid tissue in shaping the human B cell repertoire

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20122465

GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.
Figure Legend Snippet: GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.

Techniques Used: In Vivo, In Vitro, Isolation, Ex Vivo, Flow Cytometry, Cytometry, Expressing, Positive Control, FACS, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Amplification, Polymerase Chain Reaction, MANN-WHITNEY, Sequencing, Clone Assay

8) Product Images from "Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes"

Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes

Journal: Journal of Diabetes Investigation

doi: 10.1111/jdi.12063

Basal and ex vivo expression of phospho‐mitogen‐activated protein kinases ( MAPK ) in exendin‐4 treated CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and healthy subjects. (4‐1) Representative histograms show the intracellular expression of (a,b) phospho‐extracellular signal‐regulated kinase ( ERK ), (c,d) phosphor‐p38 and (e,f) phosphor‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in peripheral blood mononuclear cells incubated without or with exendin‐4 (50 nmol/L) for 10 min. (4‐2) Results are expressed in bar charts. (a) Phosphorylation of ERK , (b), phosphorylation of p38 MAPK and (c) phosphorylation of JNK in monocytes and CD 4+ T lymphocytes from patients with type2 diabetes and control subjects were analyzed by flow cytometry. Results are presented with box‐and‐whisker plots.
Figure Legend Snippet: Basal and ex vivo expression of phospho‐mitogen‐activated protein kinases ( MAPK ) in exendin‐4 treated CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and healthy subjects. (4‐1) Representative histograms show the intracellular expression of (a,b) phospho‐extracellular signal‐regulated kinase ( ERK ), (c,d) phosphor‐p38 and (e,f) phosphor‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in peripheral blood mononuclear cells incubated without or with exendin‐4 (50 nmol/L) for 10 min. (4‐2) Results are expressed in bar charts. (a) Phosphorylation of ERK , (b), phosphorylation of p38 MAPK and (c) phosphorylation of JNK in monocytes and CD 4+ T lymphocytes from patients with type2 diabetes and control subjects were analyzed by flow cytometry. Results are presented with box‐and‐whisker plots.

Techniques Used: Ex Vivo, Expressing, Incubation, Flow Cytometry, Cytometry, Whisker Assay

Representative histograms of flow cytometry analysis of phospho‐mitogen‐activated protein kinases including (a) phospho‐extracellular signal‐regulated kinase ( ERK ), (b) phospho‐p38, and (c) phospho‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and the control group ( CTL ). Triplicate experiments were carried out with essentially identical results and representative figures are shown. Immunoglobulin G 1 isotypic control antibodies, which have no specificity for target cells within a particular experiment yet retain all the non‐specific characteristics of the antibodies used in the experiment. Inclusion of this antibody is to confirm the specificity of primary antibody binding and exclude non‐specific fragment crystallizable receptor binding to cells or other cellular protein interactions.
Figure Legend Snippet: Representative histograms of flow cytometry analysis of phospho‐mitogen‐activated protein kinases including (a) phospho‐extracellular signal‐regulated kinase ( ERK ), (b) phospho‐p38, and (c) phospho‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and the control group ( CTL ). Triplicate experiments were carried out with essentially identical results and representative figures are shown. Immunoglobulin G 1 isotypic control antibodies, which have no specificity for target cells within a particular experiment yet retain all the non‐specific characteristics of the antibodies used in the experiment. Inclusion of this antibody is to confirm the specificity of primary antibody binding and exclude non‐specific fragment crystallizable receptor binding to cells or other cellular protein interactions.

Techniques Used: Flow Cytometry, Cytometry, CTL Assay, Binding Assay

9) Product Images from "c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8+ memory T-cell survival"

Article Title: c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8+ memory T-cell survival

Journal: European journal of immunology

doi: 10.1002/eji.201445342

Impaired 4-1BB-induced IκBα degradation and ERK phosphorylation in c-IAP2 H570A T cells
Figure Legend Snippet: Impaired 4-1BB-induced IκBα degradation and ERK phosphorylation in c-IAP2 H570A T cells

Techniques Used:

10) Product Images from "Role of neuritin in retinal ganglion cell death in adult mice following optic nerve injury"

Article Title: Role of neuritin in retinal ganglion cell death in adult mice following optic nerve injury

Journal: Scientific Reports

doi: 10.1038/s41598-018-28425-7

Effects of neuritin on ONI-induced activation of Akt and ERK in the retina. (A) Immunoblot analysis of phosphorylated (Phospho-) and total Akt and ERK before and 3 days after ONI in the retina of WT and neuritin KO mice. Full length blot images are presented in Supplementary Figure 3 . (B , C) Relative expression levels of phosphorylated proteins are quantified. The results are expressed as percentage of the normal WT mice and are presented as means ± S.E.M. n = 8 per group. * p
Figure Legend Snippet: Effects of neuritin on ONI-induced activation of Akt and ERK in the retina. (A) Immunoblot analysis of phosphorylated (Phospho-) and total Akt and ERK before and 3 days after ONI in the retina of WT and neuritin KO mice. Full length blot images are presented in Supplementary Figure 3 . (B , C) Relative expression levels of phosphorylated proteins are quantified. The results are expressed as percentage of the normal WT mice and are presented as means ± S.E.M. n = 8 per group. * p

Techniques Used: Activation Assay, Mouse Assay, Expressing

11) Product Images from "Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells"

Article Title: Hinokitiol Induces DNA Damage and Autophagy followed by Cell Cycle Arrest and Senescence in Gefitinib-Resistant Lung Adenocarcinoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104203

The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p
Figure Legend Snippet: The effect of hinokitiol on cell cycle distribution. H1975 cells (A) and lung stromal fibroblasts (B) were treated with 5 µM hinokitiol for 72 h. The cell cycle distribution was determined by flow cytometry after the nuclei were stained with PI. (C) BrdU incorporation assay was applied in H1975 cells treated with 5 µM hinokitiol for 72 h. (D) Western blot analysis of cyclin D1, p21, cyclin E2, cyclin A2, and cyclin B1 expression in H1975 cells. (E) Western blot analysis of EGFR and ERK expression in H1975 cells. The expression level of each protein was quantified with the NIH ImageJ program using β-actin as a loading control. (F) Abnormal mitotic morphology stained with DAPI and phalloidin were quantified at 400× magnification under a confocal microscope (TCS SP5, Leica). In (A) , (B) and ( C ), the results are representative of three different experiments, and the histogram shows the quantification expressed as the mean ± SD. *, ** and *** indicate a significant difference at the level of p

Techniques Used: Flow Cytometry, Cytometry, Staining, BrdU Incorporation Assay, Western Blot, Expressing, Microscopy

12) Product Images from "Role of neuritin in retinal ganglion cell death in adult mice following optic nerve injury"

Article Title: Role of neuritin in retinal ganglion cell death in adult mice following optic nerve injury

Journal: Scientific Reports

doi: 10.1038/s41598-018-28425-7

Effects of neuritin on ONI-induced activation of Akt and ERK in the retina. (A) . (B , C) Relative expression levels of phosphorylated proteins are quantified. The results are expressed as percentage of the normal WT mice and are presented as means ± S.E.M. n = 8 per group. * p
Figure Legend Snippet: Effects of neuritin on ONI-induced activation of Akt and ERK in the retina. (A) . (B , C) Relative expression levels of phosphorylated proteins are quantified. The results are expressed as percentage of the normal WT mice and are presented as means ± S.E.M. n = 8 per group. * p

Techniques Used: Activation Assay, Expressing, Mouse Assay

Related Articles

Flow Cytometry:

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Article Snippet: .. FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described . .. For immunofluoresence, resting and cells stimulated with anti-CD3 (5 µg/ml; 15 min) were fixed in 4% PFA, permeabilized with 0.3% saponin and stained with anti-RasGRP-1, anti-Ras, anti- SKAP-55, anti-Syntaxin 6 and appropriate fluorochrome-labelled secondary antibody, respectively.

Article Title: Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils
Article Snippet: .. Expression of phospho-ERK and phospho-p38 The expression of phospho-ERK 1/2 and phospho-p38 in human neutrophils was measured by flow cytometry using BD Phosflow (BD Biosciences) protocol for human whole blood samples. ..

Fluorescence:

Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes
Article Snippet: .. Results were expressed as mean fluorescence intensity (MFI) for the expression of intracellular phospho‐p38 MAPK, phospho‐ERK and phospho‐JNK in 10,000 cells using BD CellQuest™ software (BD FACSCalibur; BD Biosciences Corp). ..

Cytometry:

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Article Snippet: .. FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described . .. For immunofluoresence, resting and cells stimulated with anti-CD3 (5 µg/ml; 15 min) were fixed in 4% PFA, permeabilized with 0.3% saponin and stained with anti-RasGRP-1, anti-Ras, anti- SKAP-55, anti-Syntaxin 6 and appropriate fluorochrome-labelled secondary antibody, respectively.

Article Title: Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils
Article Snippet: .. Expression of phospho-ERK and phospho-p38 The expression of phospho-ERK 1/2 and phospho-p38 in human neutrophils was measured by flow cytometry using BD Phosflow (BD Biosciences) protocol for human whole blood samples. ..

Incubation:

Article Title: The FBW7-MCL-1 axis is key in M1 and M2 macrophage-related colon cancer cell progression: validating the immunotherapeutic value of targeting PI3Kγ
Article Snippet: .. These membranes were probed with SURVIVIN (#AF886, R & D Systems, Waltham, MA), BMI-1 (#sc-10745, Santa Cruz, CA, USA), Caspase-3 (#9662, Cell Signaling, MA, USA), PARP (#9542, Cell Signaling, MA, USA), E-cadherin (#610181, BD Bioscience, San Jose, CA), N-cadherin (#4061, Cell Signaling, MA, USA), VIMENTIN (#sc-32322, Santa Cruz, CA, USA), MMP2 (#13132, Cell Signaling, MA, USA), phospho AKT (#4060, Cell Signaling, MA, USA), AKT (#4691, Cell Signaling, MA, USA), phospho ERK (#612358, BD Bioscience, San Jose, CA), ERK (#9102, Cell Signaling, MA, USA), MCL-1 (#4572, Cell Signaling, MA, USA), FBW7 (#ab10752, Abcam, Cambridge, MA), and β-actin (#3700, Cell Signaling, MA, USA) primary antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA, USA). β-actin was used as a loading control for western blot analysis. .. AnimalsSix- to seven-week-old female BALB/c nu/nu and BALB/c wild-type mice weighing 15–17 g at the time of transplantation were used in this study.

FACS:

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Article Snippet: .. FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described . .. For immunofluoresence, resting and cells stimulated with anti-CD3 (5 µg/ml; 15 min) were fixed in 4% PFA, permeabilized with 0.3% saponin and stained with anti-RasGRP-1, anti-Ras, anti- SKAP-55, anti-Syntaxin 6 and appropriate fluorochrome-labelled secondary antibody, respectively.

Expressing:

Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes
Article Snippet: .. Results were expressed as mean fluorescence intensity (MFI) for the expression of intracellular phospho‐p38 MAPK, phospho‐ERK and phospho‐JNK in 10,000 cells using BD CellQuest™ software (BD FACSCalibur; BD Biosciences Corp). ..

Article Title: Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils
Article Snippet: .. Expression of phospho-ERK and phospho-p38 The expression of phospho-ERK 1/2 and phospho-p38 in human neutrophils was measured by flow cytometry using BD Phosflow (BD Biosciences) protocol for human whole blood samples. ..

Staining:

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Article Snippet: .. FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described . .. For immunofluoresence, resting and cells stimulated with anti-CD3 (5 µg/ml; 15 min) were fixed in 4% PFA, permeabilized with 0.3% saponin and stained with anti-RasGRP-1, anti-Ras, anti- SKAP-55, anti-Syntaxin 6 and appropriate fluorochrome-labelled secondary antibody, respectively.

Western Blot:

Article Title: The FBW7-MCL-1 axis is key in M1 and M2 macrophage-related colon cancer cell progression: validating the immunotherapeutic value of targeting PI3Kγ
Article Snippet: .. These membranes were probed with SURVIVIN (#AF886, R & D Systems, Waltham, MA), BMI-1 (#sc-10745, Santa Cruz, CA, USA), Caspase-3 (#9662, Cell Signaling, MA, USA), PARP (#9542, Cell Signaling, MA, USA), E-cadherin (#610181, BD Bioscience, San Jose, CA), N-cadherin (#4061, Cell Signaling, MA, USA), VIMENTIN (#sc-32322, Santa Cruz, CA, USA), MMP2 (#13132, Cell Signaling, MA, USA), phospho AKT (#4060, Cell Signaling, MA, USA), AKT (#4691, Cell Signaling, MA, USA), phospho ERK (#612358, BD Bioscience, San Jose, CA), ERK (#9102, Cell Signaling, MA, USA), MCL-1 (#4572, Cell Signaling, MA, USA), FBW7 (#ab10752, Abcam, Cambridge, MA), and β-actin (#3700, Cell Signaling, MA, USA) primary antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA, USA). β-actin was used as a loading control for western blot analysis. .. AnimalsSix- to seven-week-old female BALB/c nu/nu and BALB/c wild-type mice weighing 15–17 g at the time of transplantation were used in this study.

Immunofluorescence:

Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells
Article Snippet: .. FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described . .. For immunofluoresence, resting and cells stimulated with anti-CD3 (5 µg/ml; 15 min) were fixed in 4% PFA, permeabilized with 0.3% saponin and stained with anti-RasGRP-1, anti-Ras, anti- SKAP-55, anti-Syntaxin 6 and appropriate fluorochrome-labelled secondary antibody, respectively.

Software:

Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes
Article Snippet: .. Results were expressed as mean fluorescence intensity (MFI) for the expression of intracellular phospho‐p38 MAPK, phospho‐ERK and phospho‐JNK in 10,000 cells using BD CellQuest™ software (BD FACSCalibur; BD Biosciences Corp). ..

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    Becton Dickinson phospho erk
    SKAP-55 deficient T-cells show enhanced anti-CD3 induced <t>ERK</t> activation. Panel A: <t>FACS</t> profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .
    Phospho Erk, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti phospho erk
    Intracellular signaling in CQ-exposed T-cells. ( A ) CD4 + T-cells were activated in the presence or absence (Medium) of the indicated concentrations of CQ. After 24 hours phosphorylation of <t>S6RP,</t> p38 and <t>ERK</t> was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells (S6RP) or mean fluorescence intensity (p38 and ERK). One representative experiment (n = 6) is shown. ( B ) Jurkat T-cells expressing either an NFAT::GFP (left column), NF-κB::GFP (middle column) or AP-1::GFP (right column) reporter construct were activated with anti-CD3/CD80 expressing T-cell stimulator cells in the presence or absence (Medium) of CQ. After 24 hours GFP expression as readout for promoter activity was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells. One representative experiment is shown (n = 6).
    Anti Phospho Erk, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .

    Journal: PLoS ONE

    Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

    doi: 10.1371/journal.pone.0001718

    Figure Lengend Snippet: SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .

    Article Snippet: FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described .

    Techniques: Activation Assay, FACS, Mouse Assay, Staining, Labeling, Binding Assay

    GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.

    Journal: The Journal of Experimental Medicine

    Article Title: A role for gut-associated lymphoid tissue in shaping the human B cell repertoire

    doi: 10.1084/jem.20122465

    Figure Lengend Snippet: GALT transitional B cells are activated in vivo and in vitro in response to intestinal bacteria. (A) Mononuclear cells were isolated from Peyer’s patches and analyzed directly ex vivo by flow cytometry for their expression of CD19, CD10, IgD, and phospho-BTK, phospho-Syk, and phospho-ERK. Representative histograms displaying the degree of BTK, Syk, and ERK phosphorylation for CD19 + CD10 + IgD + transitional (TS) and CD19 + CD10 + IgD − germinal center (GC) B cells. Shown is one of three experiments with similar results. (B) PBMCs were stimulated polyclonally for 30 min with CpG, anti-IgM, and heat-inactivated intestinal bacteria as a positive control for B cell kinase phosphorylation, or left unstimulated. After stimulation, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD27, CD24, CD38, and phospho-BTK, phospho-Syk, and phospho-ERK. Histograms display the degree of BTK, Syk, and ERK phosphorylation for T1, T2, and naive B cells. Histograms show one of three experiments. (C) FACS-sorted T1 cells (CD19 + CD20 + CD27 − CD24 ++ CD38 ++ ), T2 cells (CD19 + CD20 + CD27 − CD24 + CD38 + ), and naive mature B cells (CD19 + CD20 + CD27 − CD24 − CD38 − ) isolated from normal blood were incubated with combinations of heat-inactivated intestinal bacteria, CpG, and anti-IgM for 30 min, followed by intracellular staining for phospho-BTK. Mean values + SD from three independent experiments are shows. (D and E) Autoantibody secretion by in vitro stimulated peripheral blood and Peyer’s patch B cells. T1, T2, and naive mature B cells were FACS sorted (sort strategy as in Fig. S2 A ) from normal blood and Peyer’s patches (one experiment with pooled cells from three donors) and were incubated with heat-inactivated intestinal bacteria for 8 d. Supernatants were screened for secreted autoantibodies by Hep-2 ELISA. Graphs show Hep-2 binding IgG (D) and IgA (E) autoantibodies. Data are standardized so that reactivity of supernatant from blood naive cells is 1 for each isotype. (F and G) IGHV genes were amplified by PCR from FACS-sorted TS and GC B cells isolated from Peyer’s patches of seven different donors (gated and sorted as shown in Fig. S2 B ) from two sorts. (F) Pooled data of the number of mutations in GC or TS IGHV genes. Mann-Whitney test, P = 0.0001. (G) Number of mutations in GC or TS IGHV genes segregated by donor. Horizontal bars show the mean number of mutations. (H and I) An example of mutations in clonally related germinal center B cells. (H) Examples of IGHV sequencing of germinal center B cells showing four clonally related sequences deriving from a common ancestor. (I) Graphical explanation of the relation of the clones in H to a common ancestor and their germline precursor.

    Article Snippet: Antibodies were used at concentrations recommended by the manufacturers: phospho-BTK, phospho-syk, phospho-ERK, CD79b, CD10, IgD, HLA-DR, and CD11c (all BD); CD19, CD38, CD27, CD24, and IgM (all BioLegend); CD20 efluor 450 and β7 (eBioscience); and dendritic cell exclusion (“lineage”) cocktail (anti-CD3/CD14/CD16/CD19/CD34; AbD Serotec).

    Techniques: In Vivo, In Vitro, Isolation, Ex Vivo, Flow Cytometry, Cytometry, Expressing, Positive Control, FACS, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Amplification, Polymerase Chain Reaction, MANN-WHITNEY, Sequencing, Clone Assay

    Basal and ex vivo expression of phospho‐mitogen‐activated protein kinases ( MAPK ) in exendin‐4 treated CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and healthy subjects. (4‐1) Representative histograms show the intracellular expression of (a,b) phospho‐extracellular signal‐regulated kinase ( ERK ), (c,d) phosphor‐p38 and (e,f) phosphor‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in peripheral blood mononuclear cells incubated without or with exendin‐4 (50 nmol/L) for 10 min. (4‐2) Results are expressed in bar charts. (a) Phosphorylation of ERK , (b), phosphorylation of p38 MAPK and (c) phosphorylation of JNK in monocytes and CD 4+ T lymphocytes from patients with type2 diabetes and control subjects were analyzed by flow cytometry. Results are presented with box‐and‐whisker plots.

    Journal: Journal of Diabetes Investigation

    Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes

    doi: 10.1111/jdi.12063

    Figure Lengend Snippet: Basal and ex vivo expression of phospho‐mitogen‐activated protein kinases ( MAPK ) in exendin‐4 treated CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and healthy subjects. (4‐1) Representative histograms show the intracellular expression of (a,b) phospho‐extracellular signal‐regulated kinase ( ERK ), (c,d) phosphor‐p38 and (e,f) phosphor‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in peripheral blood mononuclear cells incubated without or with exendin‐4 (50 nmol/L) for 10 min. (4‐2) Results are expressed in bar charts. (a) Phosphorylation of ERK , (b), phosphorylation of p38 MAPK and (c) phosphorylation of JNK in monocytes and CD 4+ T lymphocytes from patients with type2 diabetes and control subjects were analyzed by flow cytometry. Results are presented with box‐and‐whisker plots.

    Article Snippet: Results were expressed as mean fluorescence intensity (MFI) for the expression of intracellular phospho‐p38 MAPK, phospho‐ERK and phospho‐JNK in 10,000 cells using BD CellQuest™ software (BD FACSCalibur; BD Biosciences Corp).

    Techniques: Ex Vivo, Expressing, Incubation, Flow Cytometry, Cytometry, Whisker Assay

    Representative histograms of flow cytometry analysis of phospho‐mitogen‐activated protein kinases including (a) phospho‐extracellular signal‐regulated kinase ( ERK ), (b) phospho‐p38, and (c) phospho‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and the control group ( CTL ). Triplicate experiments were carried out with essentially identical results and representative figures are shown. Immunoglobulin G 1 isotypic control antibodies, which have no specificity for target cells within a particular experiment yet retain all the non‐specific characteristics of the antibodies used in the experiment. Inclusion of this antibody is to confirm the specificity of primary antibody binding and exclude non‐specific fragment crystallizable receptor binding to cells or other cellular protein interactions.

    Journal: Journal of Diabetes Investigation

    Article Title: Anti-inflammatory effects of exendin-4, a glucagon-like peptide-1 analog, on human peripheral lymphocytes in patients with type 2 diabetes

    doi: 10.1111/jdi.12063

    Figure Lengend Snippet: Representative histograms of flow cytometry analysis of phospho‐mitogen‐activated protein kinases including (a) phospho‐extracellular signal‐regulated kinase ( ERK ), (b) phospho‐p38, and (c) phospho‐c‐Jun NH 2‐terminal protein kinase ( JNK ) in CD 4+ T lymphocytes and monocytes from peripheral blood mononuclear cells in patients with type 2 diabetes ( T 2 DM ) and the control group ( CTL ). Triplicate experiments were carried out with essentially identical results and representative figures are shown. Immunoglobulin G 1 isotypic control antibodies, which have no specificity for target cells within a particular experiment yet retain all the non‐specific characteristics of the antibodies used in the experiment. Inclusion of this antibody is to confirm the specificity of primary antibody binding and exclude non‐specific fragment crystallizable receptor binding to cells or other cellular protein interactions.

    Article Snippet: Results were expressed as mean fluorescence intensity (MFI) for the expression of intracellular phospho‐p38 MAPK, phospho‐ERK and phospho‐JNK in 10,000 cells using BD CellQuest™ software (BD FACSCalibur; BD Biosciences Corp).

    Techniques: Flow Cytometry, Cytometry, CTL Assay, Binding Assay

    Intracellular signaling in CQ-exposed T-cells. ( A ) CD4 + T-cells were activated in the presence or absence (Medium) of the indicated concentrations of CQ. After 24 hours phosphorylation of S6RP, p38 and ERK was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells (S6RP) or mean fluorescence intensity (p38 and ERK). One representative experiment (n = 6) is shown. ( B ) Jurkat T-cells expressing either an NFAT::GFP (left column), NF-κB::GFP (middle column) or AP-1::GFP (right column) reporter construct were activated with anti-CD3/CD80 expressing T-cell stimulator cells in the presence or absence (Medium) of CQ. After 24 hours GFP expression as readout for promoter activity was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells. One representative experiment is shown (n = 6).

    Journal: Scientific Reports

    Article Title: Chloroquine inhibits human CD4+ T-cell activation by AP-1 signaling modulation

    doi: 10.1038/srep42191

    Figure Lengend Snippet: Intracellular signaling in CQ-exposed T-cells. ( A ) CD4 + T-cells were activated in the presence or absence (Medium) of the indicated concentrations of CQ. After 24 hours phosphorylation of S6RP, p38 and ERK was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells (S6RP) or mean fluorescence intensity (p38 and ERK). One representative experiment (n = 6) is shown. ( B ) Jurkat T-cells expressing either an NFAT::GFP (left column), NF-κB::GFP (middle column) or AP-1::GFP (right column) reporter construct were activated with anti-CD3/CD80 expressing T-cell stimulator cells in the presence or absence (Medium) of CQ. After 24 hours GFP expression as readout for promoter activity was measured by FACS. Black line: unstimulated cells, grey histograms: stimulated cells. Numbers indicate percentage of positive cells. One representative experiment is shown (n = 6).

    Article Snippet: Afterwards, cells were washed twice in PBS + 0.5% BSA + 0.05% NaN3 and 20 μL anti-phospho-S6RP (S240; Alexa Fluor 647 conjugated; clone N4-41), anti-phospho-ERK (T202/Y204; Pacific Blue conjugated; clone 20 A) and anti-phospho-p38 (T180/Y182; PE conjugated; clone 36/p38; BD Phosflow) or isotype-matched control antibodies were added for 60 minutes.

    Techniques: FACS, Fluorescence, Expressing, Construct, Activity Assay