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Cell Signaling Technology Inc erk1 2
Differential expression patterns of YB-1, c-Kit, MAPT and GST in time course . MCF-7 cells that incubated with 20 nM doxorubicin for the indicated periods of time revealed different kinetic patterns for YB-1 and GST expressions between sensitive and resistant MCF-7 cells. A: Dot blot array analysis on doxorubicin sensitive MCF-7 cells. B: Dot blot array analysis on the doxorubicin resistant MCF-7 cells. A and B: The scale on x-axis is not in proportion with time. In the time course study, actin was employed as a control for normalization, because GAPDH was regulated in doxorubicin resistant MCF-7 cells. C: Effect of doxorubicin on the expression of drug resistance related target proteins YB-1, c-Kit, <t>ERK1/2,</t> ERK3, FAS, MAPT, MDR1, ABCB5 and PARP-1 in the four subtypes of MCF-7 cells. 1, 2 are MCF-7 and MCF-7/vector-YB-1 respectively without treatment of doxorubicin. 3, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (not fused cells); 4, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (fused cells, FACS sorted R2); 5, doxorubicin resistant MCF-7 cell line. Numbers indicate a relative level of protein expression based on the level of intensity of β-actin after normalization.
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1) Product Images from "p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance"

Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-388

Differential expression patterns of YB-1, c-Kit, MAPT and GST in time course . MCF-7 cells that incubated with 20 nM doxorubicin for the indicated periods of time revealed different kinetic patterns for YB-1 and GST expressions between sensitive and resistant MCF-7 cells. A: Dot blot array analysis on doxorubicin sensitive MCF-7 cells. B: Dot blot array analysis on the doxorubicin resistant MCF-7 cells. A and B: The scale on x-axis is not in proportion with time. In the time course study, actin was employed as a control for normalization, because GAPDH was regulated in doxorubicin resistant MCF-7 cells. C: Effect of doxorubicin on the expression of drug resistance related target proteins YB-1, c-Kit, ERK1/2, ERK3, FAS, MAPT, MDR1, ABCB5 and PARP-1 in the four subtypes of MCF-7 cells. 1, 2 are MCF-7 and MCF-7/vector-YB-1 respectively without treatment of doxorubicin. 3, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (not fused cells); 4, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (fused cells, FACS sorted R2); 5, doxorubicin resistant MCF-7 cell line. Numbers indicate a relative level of protein expression based on the level of intensity of β-actin after normalization.
Figure Legend Snippet: Differential expression patterns of YB-1, c-Kit, MAPT and GST in time course . MCF-7 cells that incubated with 20 nM doxorubicin for the indicated periods of time revealed different kinetic patterns for YB-1 and GST expressions between sensitive and resistant MCF-7 cells. A: Dot blot array analysis on doxorubicin sensitive MCF-7 cells. B: Dot blot array analysis on the doxorubicin resistant MCF-7 cells. A and B: The scale on x-axis is not in proportion with time. In the time course study, actin was employed as a control for normalization, because GAPDH was regulated in doxorubicin resistant MCF-7 cells. C: Effect of doxorubicin on the expression of drug resistance related target proteins YB-1, c-Kit, ERK1/2, ERK3, FAS, MAPT, MDR1, ABCB5 and PARP-1 in the four subtypes of MCF-7 cells. 1, 2 are MCF-7 and MCF-7/vector-YB-1 respectively without treatment of doxorubicin. 3, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (not fused cells); 4, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (fused cells, FACS sorted R2); 5, doxorubicin resistant MCF-7 cell line. Numbers indicate a relative level of protein expression based on the level of intensity of β-actin after normalization.

Techniques Used: Expressing, Incubation, Dot Blot, Plasmid Preparation, FACS

2) Product Images from "EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice"

Article Title: EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice

Journal: Scientific Reports

doi: 10.1038/srep24580

PA induces EGFR activation through TLR4/c-Src signaling cascade in H9C2 cells. ( A–D ) EGFR siRNA attenuated PA-induced cell injury. H9C2 cells were pretreated with EGFR siRNA for 6 h, and the down-regulation of EGFR protein expression was determined by western blotting ( A ). A scrambled sequence for the EGFR siRNA was used as the negative control (si-NC). Similar results were observed in three independent experiments. After incubating for 24 h, EGFR-silencing H9C2 cells were stimulated with PA (100 μM) for 6 h. TNF-ɑ ( B ) and ANP ( C ) mRNA levels were measured by qPCR and normalized by β-actin. Bars represent the mean ± SD of four independent experiments run in triplicate. The intracellular caspase-3 activity was evaluated upon PA (100 μM) treatment for 24 h ( D ). ( E–H ) TLR4 siRNA inhibited PA-induced EGFR pathway activation. H9C2 cells were pretreated with siTLR4 for 6 h to knock down TLR4. The phosphorylation levels of EGFR ( E ), c-Src ( F ), AKT ( G ), and ERK1/2 ( H ) were determined by western blot method after PA (100 μM) treatment for 15 min. The column figure shows the normalized optical density from four independent experiments. ( I,J ) H9C2 cells were pretreated with TAK-242 (TLR4 inhibitor), or PP2 (c-Src inhibitor), or AG1478 for 1 h, followed by PA (100 μM) treatment for 15 min. Phosphorylation of EGFR ( I ) and c-Src ( J ) were determined by western blot analysis. The gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots of 7 A , E – J with indicated cropping lines are shown in Supplementary Fig. S4 ). The column figure shows the normalized optical density from four independent experiments. Values are reported as means ± SEM (n = 4, # P
Figure Legend Snippet: PA induces EGFR activation through TLR4/c-Src signaling cascade in H9C2 cells. ( A–D ) EGFR siRNA attenuated PA-induced cell injury. H9C2 cells were pretreated with EGFR siRNA for 6 h, and the down-regulation of EGFR protein expression was determined by western blotting ( A ). A scrambled sequence for the EGFR siRNA was used as the negative control (si-NC). Similar results were observed in three independent experiments. After incubating for 24 h, EGFR-silencing H9C2 cells were stimulated with PA (100 μM) for 6 h. TNF-ɑ ( B ) and ANP ( C ) mRNA levels were measured by qPCR and normalized by β-actin. Bars represent the mean ± SD of four independent experiments run in triplicate. The intracellular caspase-3 activity was evaluated upon PA (100 μM) treatment for 24 h ( D ). ( E–H ) TLR4 siRNA inhibited PA-induced EGFR pathway activation. H9C2 cells were pretreated with siTLR4 for 6 h to knock down TLR4. The phosphorylation levels of EGFR ( E ), c-Src ( F ), AKT ( G ), and ERK1/2 ( H ) were determined by western blot method after PA (100 μM) treatment for 15 min. The column figure shows the normalized optical density from four independent experiments. ( I,J ) H9C2 cells were pretreated with TAK-242 (TLR4 inhibitor), or PP2 (c-Src inhibitor), or AG1478 for 1 h, followed by PA (100 μM) treatment for 15 min. Phosphorylation of EGFR ( I ) and c-Src ( J ) were determined by western blot analysis. The gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots of 7 A , E – J with indicated cropping lines are shown in Supplementary Fig. S4 ). The column figure shows the normalized optical density from four independent experiments. Values are reported as means ± SEM (n = 4, # P

Techniques Used: Activation Assay, Expressing, Western Blot, Sequencing, Negative Control, Aqueous Normal-phase Chromatography, Real-time Polymerase Chain Reaction, Activity Assay

3) Product Images from "Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor"

Article Title: Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176715

LCA inhibits ERK phosphorylation in Jurkat T cells. (A) Western blot images and (B) Quantification results of total and phosphorylated levels of ERK1/2, P38 and JNK1/2 in response to 10 μM LCA (light grey bars) or vehicle treatment (dark grey bars) in P/I-activated or resting Jurkat T cells. (C) Western blot images and (D) Quantification results of ERK1/2 phosphorylation in response to 10 μM LCA (light grey lines) or vehicle (dark grey lines) treatment in P/I-activated Jurkat T cells. LCA, lithocholic acid; P/I, PMA/ionomycin; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P
Figure Legend Snippet: LCA inhibits ERK phosphorylation in Jurkat T cells. (A) Western blot images and (B) Quantification results of total and phosphorylated levels of ERK1/2, P38 and JNK1/2 in response to 10 μM LCA (light grey bars) or vehicle treatment (dark grey bars) in P/I-activated or resting Jurkat T cells. (C) Western blot images and (D) Quantification results of ERK1/2 phosphorylation in response to 10 μM LCA (light grey lines) or vehicle (dark grey lines) treatment in P/I-activated Jurkat T cells. LCA, lithocholic acid; P/I, PMA/ionomycin; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P

Techniques Used: Western Blot

4) Product Images from "Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy"

Article Title: Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00780

Effects of SDF-1α receptor blockade via AMD3100 followed by saxagliptin treatment on oxidative stress and podocyte EMT in cultured podocytes. (A) Western blot analysis of the PKA phosphorylation levels and their quantification after treatment with HG, HG plus AMD3100, HG with saxagliptin, and pretreatment with AMD3100 for 1–2 h followed by saxagliptin under HG conditions. (B) Western blot analysis of NOX2 and its quantification in various conditions. (C,D) The amount of ROS production in podocytes detected with a DCFH-DA assay (original magnification × 200, bars = 100 μm). (E,F) Protein expression of phosphorylated ERK1/2 assessed by Western blot analysis of cell lysates under different conditions and its quantification. (G,H) Western blot analysis of TGF-β1, desmin, nephrin, and podocin under various conditions and their quantification. One-way ANOVA followed by Tukey’s test was used to compare the difference between any two of the four groups. The data are presented as the mean ± SD ( n = 3–4 for each group, 3 replicates). † P
Figure Legend Snippet: Effects of SDF-1α receptor blockade via AMD3100 followed by saxagliptin treatment on oxidative stress and podocyte EMT in cultured podocytes. (A) Western blot analysis of the PKA phosphorylation levels and their quantification after treatment with HG, HG plus AMD3100, HG with saxagliptin, and pretreatment with AMD3100 for 1–2 h followed by saxagliptin under HG conditions. (B) Western blot analysis of NOX2 and its quantification in various conditions. (C,D) The amount of ROS production in podocytes detected with a DCFH-DA assay (original magnification × 200, bars = 100 μm). (E,F) Protein expression of phosphorylated ERK1/2 assessed by Western blot analysis of cell lysates under different conditions and its quantification. (G,H) Western blot analysis of TGF-β1, desmin, nephrin, and podocin under various conditions and their quantification. One-way ANOVA followed by Tukey’s test was used to compare the difference between any two of the four groups. The data are presented as the mean ± SD ( n = 3–4 for each group, 3 replicates). † P

Techniques Used: Cell Culture, Western Blot, DCFH-DA Assay, Expressing

Proposed mechanism of podocyte EMT inhibition by saxagliptin. Saxagliptin treatment prevents SDF-1α cleavage by inhibiting enhanced DPP-4 enzyme activity and contributes to a reduction in oxidative stress markers, including NOX2, ROS and phosphorylated ERK1/2, by activating PKA phosphorylation, which hinders podocyte EMT and attenuates albuminuria. SDF-1α receptor blockade with AMD3100 blocks the function of saxagliptin in podocyte EMT, suggesting an SDF-1α role in preventing albuminuria through inhibition of podocyte EMT in vivo and in vitro .
Figure Legend Snippet: Proposed mechanism of podocyte EMT inhibition by saxagliptin. Saxagliptin treatment prevents SDF-1α cleavage by inhibiting enhanced DPP-4 enzyme activity and contributes to a reduction in oxidative stress markers, including NOX2, ROS and phosphorylated ERK1/2, by activating PKA phosphorylation, which hinders podocyte EMT and attenuates albuminuria. SDF-1α receptor blockade with AMD3100 blocks the function of saxagliptin in podocyte EMT, suggesting an SDF-1α role in preventing albuminuria through inhibition of podocyte EMT in vivo and in vitro .

Techniques Used: Inhibition, Activity Assay, In Vivo, In Vitro

Effects of saxagliptin treatment on PKA phosphorylation and oxidative stress in HFD/STZ-induced diabetic rats. (A) Expression of PKA phosphorylation was assessed by Western blot analysis. (B) Quantification of PKA phosphorylation. (C–F) Immunohistochemistry and dihydroethidium staining for NOX2 expression (original magnification × 400, bars = 50 μm) and ROS production (original magnification × 200, bars = 50 μm) as well as their quantification of expression. (G) Representative Western blot for NOX2 expression in rats. (H) Quantification of NOX2 revealed by Western blot. (I,J) Western blot result for phosphorylation of ERK1/2 as well as its quantification. One-way ANOVA followed by Tukey’s test was used to compare the differences between any two of the three groups. The data are presented as the mean ± SD ( n = 5 for each group). ∗ P
Figure Legend Snippet: Effects of saxagliptin treatment on PKA phosphorylation and oxidative stress in HFD/STZ-induced diabetic rats. (A) Expression of PKA phosphorylation was assessed by Western blot analysis. (B) Quantification of PKA phosphorylation. (C–F) Immunohistochemistry and dihydroethidium staining for NOX2 expression (original magnification × 400, bars = 50 μm) and ROS production (original magnification × 200, bars = 50 μm) as well as their quantification of expression. (G) Representative Western blot for NOX2 expression in rats. (H) Quantification of NOX2 revealed by Western blot. (I,J) Western blot result for phosphorylation of ERK1/2 as well as its quantification. One-way ANOVA followed by Tukey’s test was used to compare the differences between any two of the three groups. The data are presented as the mean ± SD ( n = 5 for each group). ∗ P

Techniques Used: Expressing, Western Blot, Immunohistochemistry, Staining

Effects of saxagliptin treatment on oxidative stress and podocyte EMT in cultured podocytes. (A) Protein expression of PKA phosphorylation as well as its quantification respectively in NG as controls, MA group as an isoosmotic control, HG group, and HG group with saxagliptin treatment. (B) NOX2 expression and its quantification in various conditions. (C,D) The amount of ROS production in podocytes detected by DCFH-DA assay (original magnification × 200, bars = 100 μm). (E,F) The protein expression of ERK1/2 phosphorylation assessed by Western blot analysis from cell lysates in different conditions and its quantification. (G,H) Western blot analysis for TGF-β1, desmin, nephrin, and podocin in various conditions and their quantifications. One-way ANOVA followed by Tukey’s test was used to compare the differences between any two of the three groups. The data are presented as the mean ± SD ( n = 3–4 for each group, 3 replicates). ∗ P
Figure Legend Snippet: Effects of saxagliptin treatment on oxidative stress and podocyte EMT in cultured podocytes. (A) Protein expression of PKA phosphorylation as well as its quantification respectively in NG as controls, MA group as an isoosmotic control, HG group, and HG group with saxagliptin treatment. (B) NOX2 expression and its quantification in various conditions. (C,D) The amount of ROS production in podocytes detected by DCFH-DA assay (original magnification × 200, bars = 100 μm). (E,F) The protein expression of ERK1/2 phosphorylation assessed by Western blot analysis from cell lysates in different conditions and its quantification. (G,H) Western blot analysis for TGF-β1, desmin, nephrin, and podocin in various conditions and their quantifications. One-way ANOVA followed by Tukey’s test was used to compare the differences between any two of the three groups. The data are presented as the mean ± SD ( n = 3–4 for each group, 3 replicates). ∗ P

Techniques Used: Cell Culture, Expressing, DCFH-DA Assay, Western Blot

5) Product Images from "Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo"

Article Title: Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo

Journal: Oncotarget

doi: 10.18632/oncotarget.22365

miR-126-3p targeted PIK3R2 and SPRED-1 and activated the ERK1/2 and AKT signaling pathways in HSVECs ( A ) Overexpression and inhibition of miR-126-3p, respectively, promoted and inhibited PIK3R2 and SPRED-1 mRNA levels in HSVECs. ( B ) Representative western blots of PIK3R2 and SPRED-1 protein expression after transfection of miR-126-3p agomir and antagomir in HSVECs. ( C ) Western blotting analyses confirmed the downregulation and upregulation of PIK3R2 and SPRED-1 proteins in HSVECs by the miR-126-3p agomir or antagomir, respectively. ( D ) Representative western blots of the upregulation and downregulation of AKT phosphorylation and ERK1/2 phosphorylation in HSVECs by the miR-126-3p agomir or antagomir, respectively. The results are presented as the mean ± SEM from 3 independent experiments. * P
Figure Legend Snippet: miR-126-3p targeted PIK3R2 and SPRED-1 and activated the ERK1/2 and AKT signaling pathways in HSVECs ( A ) Overexpression and inhibition of miR-126-3p, respectively, promoted and inhibited PIK3R2 and SPRED-1 mRNA levels in HSVECs. ( B ) Representative western blots of PIK3R2 and SPRED-1 protein expression after transfection of miR-126-3p agomir and antagomir in HSVECs. ( C ) Western blotting analyses confirmed the downregulation and upregulation of PIK3R2 and SPRED-1 proteins in HSVECs by the miR-126-3p agomir or antagomir, respectively. ( D ) Representative western blots of the upregulation and downregulation of AKT phosphorylation and ERK1/2 phosphorylation in HSVECs by the miR-126-3p agomir or antagomir, respectively. The results are presented as the mean ± SEM from 3 independent experiments. * P

Techniques Used: Over Expression, Inhibition, Western Blot, Expressing, Transfection

6) Product Images from "Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells"

Article Title: Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1405374111

PLCδ1 suppressed the phosphorylation of ERK1/2 by E-cadherin. ( A ) Control or PLCδ1-overexpressing SW620 cells (Control-1 and -2 or PLCδ1-1 and -2) were assessed for the protein levels of phosphorylated ERK1/2, ERK1/2, and β-actin
Figure Legend Snippet: PLCδ1 suppressed the phosphorylation of ERK1/2 by E-cadherin. ( A ) Control or PLCδ1-overexpressing SW620 cells (Control-1 and -2 or PLCδ1-1 and -2) were assessed for the protein levels of phosphorylated ERK1/2, ERK1/2, and β-actin

Techniques Used:

7) Product Images from "Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development"

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development

Journal: Development (Cambridge, England)

doi: 10.1242/dev.075796

Increased cell proliferation and ERK1/2 phosphorylation by FGFs in facial process mesenchymal cells. ( A-C ) Reduced ERK1/2 phosphorylation in the NP of Wnt9b –/– mouse embryos. Immunostained images (A,B) and their quantitation (C) are presented.
Figure Legend Snippet: Increased cell proliferation and ERK1/2 phosphorylation by FGFs in facial process mesenchymal cells. ( A-C ) Reduced ERK1/2 phosphorylation in the NP of Wnt9b –/– mouse embryos. Immunostained images (A,B) and their quantitation (C) are presented.

Techniques Used: Quantitation Assay

8) Product Images from "Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells"

Article Title: Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063872

UA inactivated Akt/ERK signaling to inhibit cell proloferation. ( A, B ) , SW480 cells were treated with UA at 20µM or 40 µM for 48 h. The expression of the phosphorylated or total PI3K, Akt, mTOR, PTEN, JNK, ERK1/2 proteins was detected by Western blot. GAPDH was used as a control for sample loading. ( C, D ) , SW480 cells were treated with a PI3K/Akt-selective inhibitor LY294002 (LY, 5 µM) ( C ) or with an ERK-selective inhibitor U0126 (20 µM) ( D ) for 4 hours, and then treated with UA at 20 µM. At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control DMSO. The data are presented as the mean ± SD of three separate experiments. *, P
Figure Legend Snippet: UA inactivated Akt/ERK signaling to inhibit cell proloferation. ( A, B ) , SW480 cells were treated with UA at 20µM or 40 µM for 48 h. The expression of the phosphorylated or total PI3K, Akt, mTOR, PTEN, JNK, ERK1/2 proteins was detected by Western blot. GAPDH was used as a control for sample loading. ( C, D ) , SW480 cells were treated with a PI3K/Akt-selective inhibitor LY294002 (LY, 5 µM) ( C ) or with an ERK-selective inhibitor U0126 (20 µM) ( D ) for 4 hours, and then treated with UA at 20 µM. At 48 hours after treatment, cell viability was determined by MTT analysis. The percent cell viability in each treatment group was calculated relative to cells treated with the vehicle control DMSO. The data are presented as the mean ± SD of three separate experiments. *, P

Techniques Used: Expressing, Western Blot, MTT Assay

9) Product Images from "Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1"

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

Journal: Redox Biology

doi: 10.1016/j.redox.2013.02.001

Effect of selenite supplementation on acrolein-induced MAPK activation. Non-supplemented or selenite-supplemented HBE1 cells were treated with indicated concentrations of acrolein for 30 min, and cell lysates were analyzed for p-JNK, p-ERK1/2, and p-p38 by Western blot (A). Band densities of phosphorylated p46-JNK (B) and p56-JNK (C) were quantified relative to total JNK protein. Results are expressed as mean±SEM, n =3. *: p
Figure Legend Snippet: Effect of selenite supplementation on acrolein-induced MAPK activation. Non-supplemented or selenite-supplemented HBE1 cells were treated with indicated concentrations of acrolein for 30 min, and cell lysates were analyzed for p-JNK, p-ERK1/2, and p-p38 by Western blot (A). Band densities of phosphorylated p46-JNK (B) and p56-JNK (C) were quantified relative to total JNK protein. Results are expressed as mean±SEM, n =3. *: p

Techniques Used: Activation Assay, Western Blot

10) Product Images from "ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿"

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00656-08

The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Labeling, Northern Blot, Staining, Western Blot, Positive Control

HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Plasmid Preparation, Labeling, In Vitro, Northern Blot, Staining, Western Blot, Positive Control

Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.
Figure Legend Snippet: Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.

Techniques Used: Liquid Chromatography, Tandem Mass Spectroscopy, In Vivo, Expressing, Transfection, SDS Page, Immunoprecipitation, Staining, Mass Spectrometry

U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Over Expression, Transfection, Western Blot, Northern Blot, Staining

ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.
Figure Legend Snippet: ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.

Techniques Used: In Vivo, Immunoprecipitation, SDS Page, Western Blot

The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.
Figure Legend Snippet: The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.

Techniques Used: Transfection, Plasmid Preparation, SDS Page

Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.
Figure Legend Snippet: Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.

Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Staining, Western Blot, Silver Staining

U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Transfection, In Vitro, Plasmid Preparation, Western Blot, Northern Blot, Staining

11) Product Images from "ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿"

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00656-08

The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Labeling, Northern Blot, Staining, Western Blot, Positive Control

HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Plasmid Preparation, Labeling, In Vitro, Northern Blot, Staining, Western Blot, Positive Control

Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.
Figure Legend Snippet: Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.

Techniques Used: Liquid Chromatography, Tandem Mass Spectroscopy, In Vivo, Expressing, Transfection, SDS Page, Immunoprecipitation, Staining, Mass Spectrometry

U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Over Expression, Transfection, Western Blot, Northern Blot, Staining

ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.
Figure Legend Snippet: ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.

Techniques Used: In Vivo, Immunoprecipitation, SDS Page, Western Blot

The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.
Figure Legend Snippet: The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.

Techniques Used: Transfection, Plasmid Preparation, SDS Page

Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.
Figure Legend Snippet: Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.

Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Staining, Western Blot, Silver Staining

U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Transfection, In Vitro, Plasmid Preparation, Western Blot, Northern Blot, Staining

12) Product Images from "ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿"

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00656-08

The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Labeling, Northern Blot, Staining, Western Blot, Positive Control

HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.
Figure Legend Snippet: HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

Techniques Used: Transfection, Plasmid Preparation, Labeling, In Vitro, Northern Blot, Staining, Western Blot, Positive Control

Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.
Figure Legend Snippet: Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.

Techniques Used: Liquid Chromatography, Tandem Mass Spectroscopy, In Vivo, Expressing, Transfection, SDS Page, Immunoprecipitation, Staining, Mass Spectrometry

U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Over Expression, Transfection, Western Blot, Northern Blot, Staining

ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.
Figure Legend Snippet: ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.

Techniques Used: In Vivo, Immunoprecipitation, SDS Page, Western Blot

The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.
Figure Legend Snippet: The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.

Techniques Used: Transfection, Plasmid Preparation, SDS Page

Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.
Figure Legend Snippet: Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.

Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Staining, Western Blot, Silver Staining

U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.
Figure Legend Snippet: U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

Techniques Used: Transfection, In Vitro, Plasmid Preparation, Western Blot, Northern Blot, Staining

13) Product Images from "Estrogen modulation of the ethanol-evoked myocardial oxidative stress and dysfunction via DAPK3/Akt/ERK activation in male rats"

Article Title: Estrogen modulation of the ethanol-evoked myocardial oxidative stress and dysfunction via DAPK3/Akt/ERK activation in male rats

Journal: Toxicology and applied pharmacology

doi: 10.1016/j.taap.2015.06.015

Protein expression of phosphorylated and total ERK1/2 (panel A), Akt (panel B), and DAPK3 (panel C) in ventricular homogenates obtained from male Sprague-Dawley rats 120 min after ethanol (0.5, 1, or 1.5 g/kg); estrogen (E 2 , 1 μg/kg, i.v.) or
Figure Legend Snippet: Protein expression of phosphorylated and total ERK1/2 (panel A), Akt (panel B), and DAPK3 (panel C) in ventricular homogenates obtained from male Sprague-Dawley rats 120 min after ethanol (0.5, 1, or 1.5 g/kg); estrogen (E 2 , 1 μg/kg, i.v.) or

Techniques Used: Expressing

14) Product Images from "Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase"

Article Title: Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase

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

doi: 10.1152/ajplung.00273.2010

Effects of disruption of β 2 -AR-G i coupling and ERK1/2 activation on the AC5-transgenic airway relaxation phenotype. Mice were treated by intraperitoneal injection of PTX to disrupt β 2 -AR-G i interaction, or tracheal rings treated with the MEK inhibitor U0126 to inhibit ERK1/2 activation. PTX treatment normalized the AC5-transgenic relaxation response to that of NTG. The impaired relaxation of the AC5-transgenic airways compared with NTG was not affected by the MEK inhibitor. Values are means ± SE. Results are from 4 mice in each of the 4 groups.
Figure Legend Snippet: Effects of disruption of β 2 -AR-G i coupling and ERK1/2 activation on the AC5-transgenic airway relaxation phenotype. Mice were treated by intraperitoneal injection of PTX to disrupt β 2 -AR-G i interaction, or tracheal rings treated with the MEK inhibitor U0126 to inhibit ERK1/2 activation. PTX treatment normalized the AC5-transgenic relaxation response to that of NTG. The impaired relaxation of the AC5-transgenic airways compared with NTG was not affected by the MEK inhibitor. Values are means ± SE. Results are from 4 mice in each of the 4 groups.

Techniques Used: Activation Assay, Transgenic Assay, Mouse Assay, Injection

Protein expression screening from ASM cells derived from NTG and AC5 mice. A : Western blots probing for the indicated proteins indicate decreased phosphodiesterase type 4 (PDE4) and increased G i and phosphorylated ERK1/2 (pERK1/2) in ASM from the AC5 mice. GAPDH and Na + -K + -ATPase represent intracellular and membrane-bound control proteins, respectively. Results are representative of experiments performed with 3 NTG and 4 AC5 transgenic mouse ASM cell lines. GRK2 and GRK5, G protein-coupled receptor kinases 2 and 5, respectively; β-arr, β-arrestin. B : 125 I-cyanopindolol radioligand binding indicates decreased β 2 -adrenergic receptor (β 2 -AR) expression in AC5 ASM cells. * P
Figure Legend Snippet: Protein expression screening from ASM cells derived from NTG and AC5 mice. A : Western blots probing for the indicated proteins indicate decreased phosphodiesterase type 4 (PDE4) and increased G i and phosphorylated ERK1/2 (pERK1/2) in ASM from the AC5 mice. GAPDH and Na + -K + -ATPase represent intracellular and membrane-bound control proteins, respectively. Results are representative of experiments performed with 3 NTG and 4 AC5 transgenic mouse ASM cell lines. GRK2 and GRK5, G protein-coupled receptor kinases 2 and 5, respectively; β-arr, β-arrestin. B : 125 I-cyanopindolol radioligand binding indicates decreased β 2 -adrenergic receptor (β 2 -AR) expression in AC5 ASM cells. * P

Techniques Used: Expressing, Derivative Assay, Mouse Assay, Western Blot, Transgenic Assay, Binding Assay

15) Product Images from "Dual specificity phosphatase 15 regulates Erk activation in Schwann cells"

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

Journal: Journal of neurochemistry

doi: 10.1111/jnc.13911

Dusp15 controls Erk1/2 phosphorylation levels (A) Dusp15 levels were measured using quantitative RT-PCR in RT4 Schwann cells treated with siRNA against Dusp15. (B) Western Blot analysis of Erk1/2 phosphorylation was performed and (C) quantified in RT4 Schwann cells treated with siRNA against Dusp15. (D) Dusp15 mRNA levels were compared between S16 and RT4 Schwann cells using quantitative RT-PCR. (E) Diagram depicting guide RNA (red) target sites for CRISPR/Cas9 deletion of the Dusp15 gene. (F) Genotyping of Cas9 treated cells was determined by PCR using primers (orange pair) flanking the deleted genomic and the uncut 3′UTR region in both lines (green). (G) Dusp15 mRNA levels in WT and KO cell lines were determined by quantitative RT-PCR. (H) Erk1/2 phosphorylation was measured in WT versus KO lines and compared to total levels of Erk1/2 by western blot analysis. (I) Band signals obtained from western blot analysis were normalized to Total-Erk (T-Erk) levels. All experiments represent average values of biological triplicates. (*P
Figure Legend Snippet: Dusp15 controls Erk1/2 phosphorylation levels (A) Dusp15 levels were measured using quantitative RT-PCR in RT4 Schwann cells treated with siRNA against Dusp15. (B) Western Blot analysis of Erk1/2 phosphorylation was performed and (C) quantified in RT4 Schwann cells treated with siRNA against Dusp15. (D) Dusp15 mRNA levels were compared between S16 and RT4 Schwann cells using quantitative RT-PCR. (E) Diagram depicting guide RNA (red) target sites for CRISPR/Cas9 deletion of the Dusp15 gene. (F) Genotyping of Cas9 treated cells was determined by PCR using primers (orange pair) flanking the deleted genomic and the uncut 3′UTR region in both lines (green). (G) Dusp15 mRNA levels in WT and KO cell lines were determined by quantitative RT-PCR. (H) Erk1/2 phosphorylation was measured in WT versus KO lines and compared to total levels of Erk1/2 by western blot analysis. (I) Band signals obtained from western blot analysis were normalized to Total-Erk (T-Erk) levels. All experiments represent average values of biological triplicates. (*P

Techniques Used: Quantitative RT-PCR, Western Blot, CRISPR, Polymerase Chain Reaction

16) Product Images from "MEK inhibition suppresses cell invasion and migration in ovarian cancers with activation of ERK1/2"

Article Title: MEK inhibition suppresses cell invasion and migration in ovarian cancers with activation of ERK1/2

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm_00000093

Twist immunoreactivity in ovarian cancer tissues. (A) Intense immunoreactivity is present in the nuclei of carcinoma cells. (B) In this sample negative staining for Twist is noted. (C) Correlation between the p-ERK1/2 immunohistochemical score and the Twist immunohistochemical score in ovarian cancer tissue. The HSCORE for p-ERK1/2 significantly correlates with the Twist immunohistochemical HSCORE (r=0.370, P=0.003).
Figure Legend Snippet: Twist immunoreactivity in ovarian cancer tissues. (A) Intense immunoreactivity is present in the nuclei of carcinoma cells. (B) In this sample negative staining for Twist is noted. (C) Correlation between the p-ERK1/2 immunohistochemical score and the Twist immunohistochemical score in ovarian cancer tissue. The HSCORE for p-ERK1/2 significantly correlates with the Twist immunohistochemical HSCORE (r=0.370, P=0.003).

Techniques Used: Negative Staining, Immunohistochemistry

Western blot analysis. (A) Expression of p-ERK1/2 is undetectable in all CI-1040-treated samples. A similar amount of protein was loaded in CI-1040- and DMSO-treated (control) samples as evidenced by a similar intensity of total ERK1/2. (B) Expression of Twist is undetectable or reduced in all CI-1040-treated samples.
Figure Legend Snippet: Western blot analysis. (A) Expression of p-ERK1/2 is undetectable in all CI-1040-treated samples. A similar amount of protein was loaded in CI-1040- and DMSO-treated (control) samples as evidenced by a similar intensity of total ERK1/2. (B) Expression of Twist is undetectable or reduced in all CI-1040-treated samples.

Techniques Used: Western Blot, Expressing

Immunohistochemical staining of phosphorylated extracellular-regulated kinase (p-ERK)1/2. (A) Intense immunoreactivity is present in both the nucleus and cytoplasm in this ovarian carcinoma. (B) A case showing negative staining of p-ERK1/2. (C) Kaplan-Meier survival curve in 88 patients with ovarian carcinoma according to p-ERK1/2 expression. p-ERK1/2 expression does not correlate with shorter overall survival in patients with ovarian carcinomas.
Figure Legend Snippet: Immunohistochemical staining of phosphorylated extracellular-regulated kinase (p-ERK)1/2. (A) Intense immunoreactivity is present in both the nucleus and cytoplasm in this ovarian carcinoma. (B) A case showing negative staining of p-ERK1/2. (C) Kaplan-Meier survival curve in 88 patients with ovarian carcinoma according to p-ERK1/2 expression. p-ERK1/2 expression does not correlate with shorter overall survival in patients with ovarian carcinomas.

Techniques Used: Immunohistochemistry, Staining, Negative Staining, Expressing

The effects of a MEK inhibitor on cell migration. (A) A simulated wound was created by scraping a confluent monolayer of ES2 cells. Compared to the ES2 cells treated with DMSO, there was a significant reduction in the number of CI-1040-treated ES2 cells that migrated into the wound. (B) Inactivation of p-ERK1/2 affects cell migration in ES2 and MPSC1 (p-ERK1/2-positive) but not in KF28 (p-ERK1/2-negative) cells.
Figure Legend Snippet: The effects of a MEK inhibitor on cell migration. (A) A simulated wound was created by scraping a confluent monolayer of ES2 cells. Compared to the ES2 cells treated with DMSO, there was a significant reduction in the number of CI-1040-treated ES2 cells that migrated into the wound. (B) Inactivation of p-ERK1/2 affects cell migration in ES2 and MPSC1 (p-ERK1/2-positive) but not in KF28 (p-ERK1/2-negative) cells.

Techniques Used: Migration

17) Product Images from "Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞"

Article Title: Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M709914200

Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.
Figure Legend Snippet: Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.

Techniques Used: Migration

18) Product Images from "Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞"

Article Title: Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M709914200

Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.
Figure Legend Snippet: Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.

Techniques Used: Migration

19) Product Images from "Aconitase Regulation of Erythropoiesis Correlates with a Novel Licensing Function in Erythropoietin-Induced ERK Signaling"

Article Title: Aconitase Regulation of Erythropoiesis Correlates with a Novel Licensing Function in Erythropoietin-Induced ERK Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023850

Aconitase inhibition interferes with EPO activation of ERK signaling in a pathway-specific manner. ( A ) Steady state phosphorylation of ERK and RSK as determined by immunoblotting of whole cell lysates of cells from days 3–4 erythroid cultures treated with 50 µM FA and 10 µM U0126 as indicated (NT, untreated). ( B ) Time course of ERK1/2 and RSK phosphorylation. Cells from day 3 erythroid cultures ±50 µM FA were subjected to 3 hours of cytokine starvation followed by stimulation with 4.5 U/ml EPO for the indicated time periods. ( C ) EPO induction of multiple downstream pathways in cells treated as in panel B . ( D ) Densitometric analysis of pERK1/2:ERK1/2 and pRSK:RSK levels in five independent experiments conducted as in panel C . Presented are mean values ± SEM for relative phosphoprotein signal divided by total protein signal; * P
Figure Legend Snippet: Aconitase inhibition interferes with EPO activation of ERK signaling in a pathway-specific manner. ( A ) Steady state phosphorylation of ERK and RSK as determined by immunoblotting of whole cell lysates of cells from days 3–4 erythroid cultures treated with 50 µM FA and 10 µM U0126 as indicated (NT, untreated). ( B ) Time course of ERK1/2 and RSK phosphorylation. Cells from day 3 erythroid cultures ±50 µM FA were subjected to 3 hours of cytokine starvation followed by stimulation with 4.5 U/ml EPO for the indicated time periods. ( C ) EPO induction of multiple downstream pathways in cells treated as in panel B . ( D ) Densitometric analysis of pERK1/2:ERK1/2 and pRSK:RSK levels in five independent experiments conducted as in panel C . Presented are mean values ± SEM for relative phosphoprotein signal divided by total protein signal; * P

Techniques Used: Inhibition, Activation Assay

20) Product Images from "Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-?B pathway"

Article Title: Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-?B pathway

Journal: Respiratory Research

doi: 10.1186/1465-9921-13-88

Effects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal - regulated kinase (ERK) 1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected ( A , top panel ). ERK2 was detected as the internal standard ( bottom panel ). These immunorelated protein bands were quantified and analyzed ( B ). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p
Figure Legend Snippet: Effects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal - regulated kinase (ERK) 1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected ( A , top panel ). ERK2 was detected as the internal standard ( bottom panel ). These immunorelated protein bands were quantified and analyzed ( B ). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p

Techniques Used:

21) Product Images from "ERK1/2-Egr-1 Signaling Pathway-Mediated Protective Effects of Electroacupuncture in a Mouse Model of Myocardial Ischemia-Reperfusion"

Article Title: ERK1/2-Egr-1 Signaling Pathway-Mediated Protective Effects of Electroacupuncture in a Mouse Model of Myocardial Ischemia-Reperfusion

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2014/253075

ERK1/2 activation is responsible for Egr-1expression during myocardial I/R injury. Mice received U0126 (an inhibitor of ERK1/2 kinase, 20 mg/kg, i.p.) or its vehicle 0.1% v/v DMSO treatment before surgery. As described previously, myocardial expression of Egr-1 and p-ERK1/2 was measured using western blot (a). The corresponding densitometric analysis is shown in (b-c) ( n = 3/group). The mRNA levels of Egr-1 are shown as fold increase versus U0126 + IR (d, n = 3/group). Immunohistochemical staining of Egr-1 was performed and the quantitation results of Egr-1positive cells were shown in panel (e). Representative images were shown in panel (f) (3 mice /group). Pink arrows indicate Egr-1positive cells. Scale bar = 50 μ m. * P
Figure Legend Snippet: ERK1/2 activation is responsible for Egr-1expression during myocardial I/R injury. Mice received U0126 (an inhibitor of ERK1/2 kinase, 20 mg/kg, i.p.) or its vehicle 0.1% v/v DMSO treatment before surgery. As described previously, myocardial expression of Egr-1 and p-ERK1/2 was measured using western blot (a). The corresponding densitometric analysis is shown in (b-c) ( n = 3/group). The mRNA levels of Egr-1 are shown as fold increase versus U0126 + IR (d, n = 3/group). Immunohistochemical staining of Egr-1 was performed and the quantitation results of Egr-1positive cells were shown in panel (e). Representative images were shown in panel (f) (3 mice /group). Pink arrows indicate Egr-1positive cells. Scale bar = 50 μ m. * P

Techniques Used: Activation Assay, Mouse Assay, Expressing, Western Blot, Immunohistochemistry, Staining, Quantitation Assay

Inhibiting ERK1/2 activation with U0126 protected the myocardium against I/R injury. After 24 h of reperfusion, Evans blue/TTC staining was applied to measure the infarct size ((a-b) n = 6/group). AAR/LV: area at risk/left ventricle area; IA/AAR: infarct area/area at risk. After 3 h of reperfusion, the serum cTnI level (c, n = 6/group) and the myocardial levels of TNF- α and IL-1 β ((d-e) n = 3/group) were determined using ELISA. * P
Figure Legend Snippet: Inhibiting ERK1/2 activation with U0126 protected the myocardium against I/R injury. After 24 h of reperfusion, Evans blue/TTC staining was applied to measure the infarct size ((a-b) n = 6/group). AAR/LV: area at risk/left ventricle area; IA/AAR: infarct area/area at risk. After 3 h of reperfusion, the serum cTnI level (c, n = 6/group) and the myocardial levels of TNF- α and IL-1 β ((d-e) n = 3/group) were determined using ELISA. * P

Techniques Used: Activation Assay, Staining, IA, Enzyme-linked Immunosorbent Assay

EA inhibited Egr-1 expression and ERK1/2 activation in myocardium undergoing myocardial I/R. Mice were divided into 3 groups: SHAM, IR (myocardial I/R), and EA + IR (EA stimulation was performed 30 min before myocardial I/R surgery and lasted for 30 min). After 3 h of reperfusion, the animals were sacrificed and the protein levels of Egr-1and p-ERK1/2 were measured by western blot (a) and densitometric analysis is shown in panel (b-c) ( n = 3/group). The mRNA levels of Egr-1 in these three groups are represented as the relative fold increase versus sham controls (d, n = 3/group). Immunohistochemical staining of Egr-1 was performed and the quantitation results of Egr-1positive cells were shown in panel (e). Representative images were shown in panel (f). Pink arrows indicate Egr-1positive cells. Scale bar = 50 μ m. * P
Figure Legend Snippet: EA inhibited Egr-1 expression and ERK1/2 activation in myocardium undergoing myocardial I/R. Mice were divided into 3 groups: SHAM, IR (myocardial I/R), and EA + IR (EA stimulation was performed 30 min before myocardial I/R surgery and lasted for 30 min). After 3 h of reperfusion, the animals were sacrificed and the protein levels of Egr-1and p-ERK1/2 were measured by western blot (a) and densitometric analysis is shown in panel (b-c) ( n = 3/group). The mRNA levels of Egr-1 in these three groups are represented as the relative fold increase versus sham controls (d, n = 3/group). Immunohistochemical staining of Egr-1 was performed and the quantitation results of Egr-1positive cells were shown in panel (e). Representative images were shown in panel (f). Pink arrows indicate Egr-1positive cells. Scale bar = 50 μ m. * P

Techniques Used: Expressing, Activation Assay, Mouse Assay, Western Blot, Immunohistochemistry, Staining, Quantitation Assay

Comparison of the myocardial Egr-1and p-ERK1/2 expression at different time points after I/R. The protein levels of Egr-1and p-ERK1/2 at varying reperfusion time points (R0, R3, R6, and R24) were determined by western blot (a) and the corresponding densitometric analysis is shown in (b, c). Besides, the mRNA levels of Egr-1 were measured by qRT-PCR with data presented as relative fold increase versus sham control (d). For Egr-1 and p-ERK1/2, nearly all time points exhibit significant increase comparing with sham controls and peak levels are observed at R3 time point ( P
Figure Legend Snippet: Comparison of the myocardial Egr-1and p-ERK1/2 expression at different time points after I/R. The protein levels of Egr-1and p-ERK1/2 at varying reperfusion time points (R0, R3, R6, and R24) were determined by western blot (a) and the corresponding densitometric analysis is shown in (b, c). Besides, the mRNA levels of Egr-1 were measured by qRT-PCR with data presented as relative fold increase versus sham control (d). For Egr-1 and p-ERK1/2, nearly all time points exhibit significant increase comparing with sham controls and peak levels are observed at R3 time point ( P

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

Combination of EA with ERK1/2 inhibitor did not produce more protection against myocardial I/R injury. Combination of EA and U0126 treatment was conducted to investigate the additive effects on ERK1/2/Egr-1 downregulation or the cardiac protective role. Western blot bands and corresponding densitometric analysis of Egr-1 and p-ERK1/2 are shown in (a–c) ( n = 3/group). The myocardial levels of TNF- α and IL-1 β ((d-e), n = 3/group) as well as the serum level of cTnI (f, n = 6/group) were determined.
Figure Legend Snippet: Combination of EA with ERK1/2 inhibitor did not produce more protection against myocardial I/R injury. Combination of EA and U0126 treatment was conducted to investigate the additive effects on ERK1/2/Egr-1 downregulation or the cardiac protective role. Western blot bands and corresponding densitometric analysis of Egr-1 and p-ERK1/2 are shown in (a–c) ( n = 3/group). The myocardial levels of TNF- α and IL-1 β ((d-e), n = 3/group) as well as the serum level of cTnI (f, n = 6/group) were determined.

Techniques Used: Western Blot

22) Product Images from "NFAT Targets Signaling Molecules to Gene Promoters in Pancreatic β-Cells"

Article Title: NFAT Targets Signaling Molecules to Gene Promoters in Pancreatic β-Cells

Journal: Molecular Endocrinology

doi: 10.1210/me.2014-1066

Sustained association of NFAT and ERK with the insulin gene promoter requires CN and ERK1/2 activity. ChIP-qPCR time-course analysis of fold enrichment of NFATc2 and ERK1/2 upon the insulin gene promoter in (A) MIN6 cells and (B) human islets in response
Figure Legend Snippet: Sustained association of NFAT and ERK with the insulin gene promoter requires CN and ERK1/2 activity. ChIP-qPCR time-course analysis of fold enrichment of NFATc2 and ERK1/2 upon the insulin gene promoter in (A) MIN6 cells and (B) human islets in response

Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

CN/NFAT signaling is required for ERK1/2 to translocate to the insulin gene promoter in response to glucose and GLP-1. A, Effect of shRNA targeting of NFAT family isoforms on insulin gene promoter activity in response to high (G 16.7mM) glucose and 20nM
Figure Legend Snippet: CN/NFAT signaling is required for ERK1/2 to translocate to the insulin gene promoter in response to glucose and GLP-1. A, Effect of shRNA targeting of NFAT family isoforms on insulin gene promoter activity in response to high (G 16.7mM) glucose and 20nM

Techniques Used: shRNA, Activity Assay

Schematic model for NFAT-mediated activation and repression of insulin and TNF-α genes in β-cells in response to GLP-1 and IL-1β. GLP-1 in the presence of glucose activates CN/NFAT and ERK1/2 to induce enrichment of ERK-p300 and
Figure Legend Snippet: Schematic model for NFAT-mediated activation and repression of insulin and TNF-α genes in β-cells in response to GLP-1 and IL-1β. GLP-1 in the presence of glucose activates CN/NFAT and ERK1/2 to induce enrichment of ERK-p300 and

Techniques Used: Activation Assay

23) Product Images from "Active immunization with Tocilizumab mimotopes induces specific immune responses"

Article Title: Active immunization with Tocilizumab mimotopes induces specific immune responses

Journal: BMC Biotechnology

doi: 10.1186/s12896-015-0161-9

Signaling pathway analysis with mimotope-induced antibodies. We detected the levels of phosphorylated-STAT3, STAT3, phosphorylated-Erk1/2 and Erk1/2 in HeLa, Jurkat and U937 cells to observe whether IL-6 pathway could be down-regulated by when added sera from mice immunized with mimotopes. Cells were grown at 37 °C with medium containing 10 % FBS. Two days later, the medium was replaced by FBS-free medium with sera from mimotopes immunized mice with a dilution 1:100 or Tocilizumab (10 μg/ml) at 37 °C for 2 h. Then the cells were incubated with human IL-6 (40 ng/ml) for 15 min at 37 °C. In HeLa cells ( a ), incubation with sera from mice immunized with 4A125 and 4A126 could partly reduce the levels of phosphorylated-STAT3 and phosphorylated-Erk1/2, as compared to the control group. In Jurkat cells ( b ), incubation with sera from mice immunized with 4A220 and 4A126-KLH conjugate could reduce the level of STAT3, phosphorylated-Erk1/2 and Erk1/2. In U937 cells ( c ), incubation with sera from mice immunized with 4A124, 4a125 and 4A126-KLH conjugate could increase the level of phosphorylated- STAT3, STAT3 and phosphorylated-Erk1/2
Figure Legend Snippet: Signaling pathway analysis with mimotope-induced antibodies. We detected the levels of phosphorylated-STAT3, STAT3, phosphorylated-Erk1/2 and Erk1/2 in HeLa, Jurkat and U937 cells to observe whether IL-6 pathway could be down-regulated by when added sera from mice immunized with mimotopes. Cells were grown at 37 °C with medium containing 10 % FBS. Two days later, the medium was replaced by FBS-free medium with sera from mimotopes immunized mice with a dilution 1:100 or Tocilizumab (10 μg/ml) at 37 °C for 2 h. Then the cells were incubated with human IL-6 (40 ng/ml) for 15 min at 37 °C. In HeLa cells ( a ), incubation with sera from mice immunized with 4A125 and 4A126 could partly reduce the levels of phosphorylated-STAT3 and phosphorylated-Erk1/2, as compared to the control group. In Jurkat cells ( b ), incubation with sera from mice immunized with 4A220 and 4A126-KLH conjugate could reduce the level of STAT3, phosphorylated-Erk1/2 and Erk1/2. In U937 cells ( c ), incubation with sera from mice immunized with 4A124, 4a125 and 4A126-KLH conjugate could increase the level of phosphorylated- STAT3, STAT3 and phosphorylated-Erk1/2

Techniques Used: Mouse Assay, Incubation

24) Product Images from "Colitis Induces Calcitonin Gene-related Peptide Expression and Akt Activation in Rat Primary Afferent Pathways"

Article Title: Colitis Induces Calcitonin Gene-related Peptide Expression and Akt Activation in Rat Primary Afferent Pathways

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2009.04.026

Effects of CGRP on spinal ERK1/2 and Akt phosphorylation
Figure Legend Snippet: Effects of CGRP on spinal ERK1/2 and Akt phosphorylation

Techniques Used:

25) Product Images from "Colitis Induces Calcitonin Gene-related Peptide Expression and Akt Activation in Rat Primary Afferent Pathways"

Article Title: Colitis Induces Calcitonin Gene-related Peptide Expression and Akt Activation in Rat Primary Afferent Pathways

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2009.04.026

Effects of CGRP on spinal ERK1/2 and Akt phosphorylation
Figure Legend Snippet: Effects of CGRP on spinal ERK1/2 and Akt phosphorylation

Techniques Used:

26) Product Images from "The Activation of M1 Muscarinic Receptor Signaling Induces Neuronal Differentiation in Pyramidal Hippocampal Neurons S⃞"

Article Title: The Activation of M1 Muscarinic Receptor Signaling Induces Neuronal Differentiation in Pyramidal Hippocampal Neurons S⃞

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.108.150128

Effect of carbachol on ERK1/2 activation. Hippocampal neurons were treated for 30 min in the presence of carbachol (100 μM); proteins were then extracted and analyzed by Western blot as described under Materials and Methods . A, optical densities
Figure Legend Snippet: Effect of carbachol on ERK1/2 activation. Hippocampal neurons were treated for 30 min in the presence of carbachol (100 μM); proteins were then extracted and analyzed by Western blot as described under Materials and Methods . A, optical densities

Techniques Used: Activation Assay, Western Blot

Role of ERK1/2 on carbachol-induced axonal growth in hippocampal neurons. Hippocampal neurons plated on coverslips were treated with the ERK1/2 signaling pathway inhibitors U0126 or PD98059 (A) for 24 h. Cells were then fixed and stained with a β-tubulin
Figure Legend Snippet: Role of ERK1/2 on carbachol-induced axonal growth in hippocampal neurons. Hippocampal neurons plated on coverslips were treated with the ERK1/2 signaling pathway inhibitors U0126 or PD98059 (A) for 24 h. Cells were then fixed and stained with a β-tubulin

Techniques Used: Staining

27) Product Images from "Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation"

Article Title: Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2015-0076

Propranolol dose dependently decreased cAMP levels and activated ERK1/2 in hemangioma stem cells (HemSCs). (A): HemSCs were treated with increasing doses of propranolol over a 7-log dose range (10 −11 M to 10 −5 M) and cAMP levels determined. Data presented as the fold-difference between propranolol-treated HemSCs relative to vehicle-treated HemSCs ± SEM; ∗, p
Figure Legend Snippet: Propranolol dose dependently decreased cAMP levels and activated ERK1/2 in hemangioma stem cells (HemSCs). (A): HemSCs were treated with increasing doses of propranolol over a 7-log dose range (10 −11 M to 10 −5 M) and cAMP levels determined. Data presented as the fold-difference between propranolol-treated HemSCs relative to vehicle-treated HemSCs ± SEM; ∗, p

Techniques Used:

Propranolol reduced vessel caliber and increased ERK1/2 phosphorylation in an infantile hemangioma (IH) mouse model. Hemangioma stem cell (HemSC) Matrigel implants were xenografted into the flanks of immunocompromised mice, which were split into two treatment groups: vehicle and propranolol for 3 weeks ( n = 2 cell populations; four Matrigel implants per treatment group). (A): Ultrasound-guided Doppler of implants at 21 days after implantation demonstrated reduced blood flow (red; white arrows) in implants from the propranolol treatment group compared with vehicle. (B): H E of implant sections at 21 days after implantation. Black arrows highlight blood vessels. (C): Quantification of average blood vessel number per high-power field (HPF) in implants from vehicle and propranolol treatment. (D): Quantification of average blood vessel diameter in propranolol-treated group compared with vehicle for both H49 and H50. ∗, p
Figure Legend Snippet: Propranolol reduced vessel caliber and increased ERK1/2 phosphorylation in an infantile hemangioma (IH) mouse model. Hemangioma stem cell (HemSC) Matrigel implants were xenografted into the flanks of immunocompromised mice, which were split into two treatment groups: vehicle and propranolol for 3 weeks ( n = 2 cell populations; four Matrigel implants per treatment group). (A): Ultrasound-guided Doppler of implants at 21 days after implantation demonstrated reduced blood flow (red; white arrows) in implants from the propranolol treatment group compared with vehicle. (B): H E of implant sections at 21 days after implantation. Black arrows highlight blood vessels. (C): Quantification of average blood vessel number per high-power field (HPF) in implants from vehicle and propranolol treatment. (D): Quantification of average blood vessel diameter in propranolol-treated group compared with vehicle for both H49 and H50. ∗, p

Techniques Used: Mouse Assay, Flow Cytometry

β2-Adrenergic receptor inhibition mirrored propranolol’s effects on hemangioma stem cell (HemSC) viability and ERK1/2 activation. (A): HemSCs were treated with increasing doses of atenolol, ICI, or propranolol (10 −6.5 M to 10 −3 M), and HemSC viability was assessed by Digital Imaging Microscopy System (DIMSCAN) assay at 24 hours. Data presented as survival fraction of propranolol-treated HemSCs relative to vehicle controls ± SEM. At doses of 200 μM propranolol or greater, HemSC viability was significantly greater for cells treated with atenolol compared with those treated with either ICI or propranolol ( p
Figure Legend Snippet: β2-Adrenergic receptor inhibition mirrored propranolol’s effects on hemangioma stem cell (HemSC) viability and ERK1/2 activation. (A): HemSCs were treated with increasing doses of atenolol, ICI, or propranolol (10 −6.5 M to 10 −3 M), and HemSC viability was assessed by Digital Imaging Microscopy System (DIMSCAN) assay at 24 hours. Data presented as survival fraction of propranolol-treated HemSCs relative to vehicle controls ± SEM. At doses of 200 μM propranolol or greater, HemSC viability was significantly greater for cells treated with atenolol compared with those treated with either ICI or propranolol ( p

Techniques Used: Inhibition, Activation Assay, Imaging, Microscopy

28) Product Images from "By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility, invasion and lung colonization"

Article Title: By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility, invasion and lung colonization

Journal: Oncotarget

doi:

MMP-7 enhances the migration and invasion of human chondrosarcoma cells SW1353 cells were treated with forskolin (10 μM) (A, B) , or IL-1β (100 ng/ml) (C, D) in the absence or presence of PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM), JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM). In select experiments, SW1353 cells were exposed to either static or shear (2 dyn/cm 2 ) for 48 h. Cells suspended in their corresponding conditioned medium were then subjected to transwell experimens (E, F) . In separate experiments, SW1353 cells were incubated with rhMMP-7 (1 μg/ml) or vehicle control before being seeded to the transwell device (G, H) . In distinct experiments, SW1353 cells were transfected with either MMP-7 cDNA or the empty vector before being seeded to transwell device (I, J) . * p
Figure Legend Snippet: MMP-7 enhances the migration and invasion of human chondrosarcoma cells SW1353 cells were treated with forskolin (10 μM) (A, B) , or IL-1β (100 ng/ml) (C, D) in the absence or presence of PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM), JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM). In select experiments, SW1353 cells were exposed to either static or shear (2 dyn/cm 2 ) for 48 h. Cells suspended in their corresponding conditioned medium were then subjected to transwell experimens (E, F) . In separate experiments, SW1353 cells were incubated with rhMMP-7 (1 μg/ml) or vehicle control before being seeded to the transwell device (G, H) . In distinct experiments, SW1353 cells were transfected with either MMP-7 cDNA or the empty vector before being seeded to transwell device (I, J) . * p

Techniques Used: Migration, Incubation, Transfection, Plasmid Preparation

Signaling cascades involved in MMP-7 regulation in response to fluid shear stress, which in turn mediates chondrosarcoma metastasis Fluid shear stress (2 dyn/cm 2 ) induces cAMP and IL-1β production and release in human SW1353 chondrosarcoma cells. Elevated levels of cAMP and IL-1β stimulate the activity of PI3-K/AKT, p38 and ERK1/2 pathways, which in turn upregulate MMP-7 synthesis in a c-Jun- and NF-κB-dependent manner. Transactivation of MMP-7 mediates chondrosarcoma migration and invasion, thereby contributing to chondrosarcoma metastasis.
Figure Legend Snippet: Signaling cascades involved in MMP-7 regulation in response to fluid shear stress, which in turn mediates chondrosarcoma metastasis Fluid shear stress (2 dyn/cm 2 ) induces cAMP and IL-1β production and release in human SW1353 chondrosarcoma cells. Elevated levels of cAMP and IL-1β stimulate the activity of PI3-K/AKT, p38 and ERK1/2 pathways, which in turn upregulate MMP-7 synthesis in a c-Jun- and NF-κB-dependent manner. Transactivation of MMP-7 mediates chondrosarcoma migration and invasion, thereby contributing to chondrosarcoma metastasis.

Techniques Used: Activity Assay, Migration

Involvement of cAMP and IL-1β in mediating fluid shear stress to regulate the synthesis of MMP-7 via AKT, ERK1/2 and p38 signaling pathways in SW1353 cells SW1353 cells were subjected to fluid shear stress (2 dyn/cm 2 ) or static conditions (0 dyn/cm 2 ) in the absence or presence of adenylyl cyclase inhibitor, SQ22536 (10 μM) or IL-1β mAb (1 μg/ml) for 48 h (A) In select experiments, SW1353 cells were exposed in shear stress (2 dyn/cm 2 ) (B) , forskolin (10 μM) (D) or IL-1β (100 ng/ml) (E) in the absence or presence of LY294002 (10 μM), SB203580 (10 μM) or U0126 (10 μM) for 48 h. In separate experiments, SW1353 cells were transfected with siRNAs targeted to AKT, p38 or ERK1/2 before subjecting to fluid shear stress (2 dyn/cm 2 ) (C) Phosphorylated AKT, ERK1/2 and p38 are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total AKT, ERK1/2, p38 and β-actin. These western blots are representative of three independent experiments, all revealing similar results. MMP-7 mRNA and protein levels were determined by qRT-PCR and zymography, respectively. GAPDH and MMP-11 total protein amount served for internal control in qRT-PCR and zymography assays, respectively. Data represent the mean ± S.E. of 3 independent experiments. * p
Figure Legend Snippet: Involvement of cAMP and IL-1β in mediating fluid shear stress to regulate the synthesis of MMP-7 via AKT, ERK1/2 and p38 signaling pathways in SW1353 cells SW1353 cells were subjected to fluid shear stress (2 dyn/cm 2 ) or static conditions (0 dyn/cm 2 ) in the absence or presence of adenylyl cyclase inhibitor, SQ22536 (10 μM) or IL-1β mAb (1 μg/ml) for 48 h (A) In select experiments, SW1353 cells were exposed in shear stress (2 dyn/cm 2 ) (B) , forskolin (10 μM) (D) or IL-1β (100 ng/ml) (E) in the absence or presence of LY294002 (10 μM), SB203580 (10 μM) or U0126 (10 μM) for 48 h. In separate experiments, SW1353 cells were transfected with siRNAs targeted to AKT, p38 or ERK1/2 before subjecting to fluid shear stress (2 dyn/cm 2 ) (C) Phosphorylated AKT, ERK1/2 and p38 are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total AKT, ERK1/2, p38 and β-actin. These western blots are representative of three independent experiments, all revealing similar results. MMP-7 mRNA and protein levels were determined by qRT-PCR and zymography, respectively. GAPDH and MMP-11 total protein amount served for internal control in qRT-PCR and zymography assays, respectively. Data represent the mean ± S.E. of 3 independent experiments. * p

Techniques Used: Transfection, Western Blot, Quantitative RT-PCR, Zymography

Fluid shear stress activates transcriptional factors, c-Jun and NF-κB, via PI3-K, p38 and ERK1/2 pathways, which result in MMP-7 induction in human SW1353 chondrosarcoma cells SW1353 cells were subjected to fluid shear stress (2 dyn/cm 2 ) or static conditions (0 dyn/cm 2 ) for 48 h in the absence or presence of either adenylyl cyclase inhibitor, SQ22536 (10 μM), IL-1β mAb (1 μg/ml) (A) , PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM) (B) JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM) for 48 h (E) . In select experiments, SW1353 cells were treated with 10 μM of forskolin (C, F) or 100 ng/ml of IL-1β (D, G) in the absence or presence of PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM) (B, D) JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM) for 48 h (F, G). The levels of phosphor-c-Jun, phosphor-NF-κB and total levels of c-Jun, NF-κB from cell lysates were determined by western blots using specific Abs. Equal loading in each lane is ensured by the similar intensities of total β-actin. The gels are representative of at least three independent experiments, all revealing similar results (A-G) MMP-7 mRNA and protein levels were determined by qRT-PCR and zymography, respectively. GAPDH and MMP-11 total protein amount served for internal control in qRT-PCR and Zymography assays, respectively (E-G) Data represent the mean ± S.E. of 3 independent experiments. * p
Figure Legend Snippet: Fluid shear stress activates transcriptional factors, c-Jun and NF-κB, via PI3-K, p38 and ERK1/2 pathways, which result in MMP-7 induction in human SW1353 chondrosarcoma cells SW1353 cells were subjected to fluid shear stress (2 dyn/cm 2 ) or static conditions (0 dyn/cm 2 ) for 48 h in the absence or presence of either adenylyl cyclase inhibitor, SQ22536 (10 μM), IL-1β mAb (1 μg/ml) (A) , PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM) (B) JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM) for 48 h (E) . In select experiments, SW1353 cells were treated with 10 μM of forskolin (C, F) or 100 ng/ml of IL-1β (D, G) in the absence or presence of PI3-K inhibitor, LY294002 (10 μM), p38 inhibitor, SB203580 (10 μM), ERK1/2 inhibitor, U0126 (10 μM) (B, D) JNK inhibitor, SP600125 (10 μM) or NF-κB inhibitor, QNZ (2 μM) for 48 h (F, G). The levels of phosphor-c-Jun, phosphor-NF-κB and total levels of c-Jun, NF-κB from cell lysates were determined by western blots using specific Abs. Equal loading in each lane is ensured by the similar intensities of total β-actin. The gels are representative of at least three independent experiments, all revealing similar results (A-G) MMP-7 mRNA and protein levels were determined by qRT-PCR and zymography, respectively. GAPDH and MMP-11 total protein amount served for internal control in qRT-PCR and Zymography assays, respectively (E-G) Data represent the mean ± S.E. of 3 independent experiments. * p

Techniques Used: Western Blot, Quantitative RT-PCR, Zymography

29) Product Images from "Epidermal growth factor receptor inhibits colitis-associated cancer in mice"

Article Title: Epidermal growth factor receptor inhibits colitis-associated cancer in mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI62888

Mutually exclusive status of ERK1/2 phosphorylation and nuclear β-catenin in different mouse colon tumor models.
Figure Legend Snippet: Mutually exclusive status of ERK1/2 phosphorylation and nuclear β-catenin in different mouse colon tumor models.

Techniques Used:

30) Product Images from "Hyaluronan Stabilizes Focal Adhesions, Filopodia, and the Proliferative Phenotype in Esophageal Squamous Carcinoma Cells *"

Article Title: Hyaluronan Stabilizes Focal Adhesions, Filopodia, and the Proliferative Phenotype in Esophageal Squamous Carcinoma Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.093146

4-MU-induced FAK cleavage coincides with decreased phosphorylation of ERK1/2 and reduced proliferation and migration. A , OSC1 cells were incubated with 4-MU (0.3 m m ), and FAK degradation was detected with an antibody targeting the C terminus of tFAK starting
Figure Legend Snippet: 4-MU-induced FAK cleavage coincides with decreased phosphorylation of ERK1/2 and reduced proliferation and migration. A , OSC1 cells were incubated with 4-MU (0.3 m m ), and FAK degradation was detected with an antibody targeting the C terminus of tFAK starting

Techniques Used: Migration, Incubation

31) Product Images from "BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells"

Article Title: BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2012-0051OC

SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then
Figure Legend Snippet: SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then

Techniques Used:

Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in
Figure Legend Snippet: Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in

Techniques Used: Expressing, Activation Assay, Binding Assay

SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)
Figure Legend Snippet: SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)

Techniques Used: Expressing

Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4
Figure Legend Snippet: Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4

Techniques Used: Expressing, Incubation

Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells
Figure Legend Snippet: Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells

Techniques Used:

Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5
Figure Legend Snippet: Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5

Techniques Used: Inhibition, Activation Assay, Expressing

32) Product Images from "BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells"

Article Title: BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2012-0051OC

SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then
Figure Legend Snippet: SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then

Techniques Used:

Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in
Figure Legend Snippet: Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in

Techniques Used: Expressing, Activation Assay, Binding Assay

SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)
Figure Legend Snippet: SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)

Techniques Used: Expressing

Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4
Figure Legend Snippet: Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4

Techniques Used: Expressing, Incubation

Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells
Figure Legend Snippet: Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells

Techniques Used:

Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5
Figure Legend Snippet: Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5

Techniques Used: Inhibition, Activation Assay, Expressing

33) Product Images from "BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells"

Article Title: BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2012-0051OC

SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then
Figure Legend Snippet: SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then

Techniques Used:

Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in
Figure Legend Snippet: Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in

Techniques Used: Expressing, Activation Assay, Binding Assay

SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)
Figure Legend Snippet: SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM)

Techniques Used: Expressing

Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4
Figure Legend Snippet: Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4

Techniques Used: Expressing, Incubation

Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells
Figure Legend Snippet: Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells

Techniques Used:

Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5
Figure Legend Snippet: Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca 2+ ([Ca 2+ ] i ) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5

Techniques Used: Inhibition, Activation Assay, Expressing

34) Product Images from "Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells"

Article Title: Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-10-15

Hon inhibits IFNγ-induced activation of p-ERK1/2 in HAPI microglial cells. (A) Western blot analysis showing a representative experiment of Hon pretreatment on IFNγ to induce ERK1/2 phosphorylation in HAPI microglial cells. Cells were treated with Hon (1 to 10 μM) for 1 h followed by stimulation with IFNγ (10 ng/ml) for 4 h. (B C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2. Results are expressed as the mean ± SEM ( n = 3) and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, * P
Figure Legend Snippet: Hon inhibits IFNγ-induced activation of p-ERK1/2 in HAPI microglial cells. (A) Western blot analysis showing a representative experiment of Hon pretreatment on IFNγ to induce ERK1/2 phosphorylation in HAPI microglial cells. Cells were treated with Hon (1 to 10 μM) for 1 h followed by stimulation with IFNγ (10 ng/ml) for 4 h. (B C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2. Results are expressed as the mean ± SEM ( n = 3) and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, * P

Techniques Used: Activation Assay, Western Blot

Time course of IFNγ-induced activation of p-ERK1/2 in BV-2 microglial cells. (A) Western blot analysis of a typical time course for IFNγ (10 ng/ml) to induce ERK1/2 phosphorylation in BV-2 microglia cells. Cell lysates were extracted at the time indicated. (B, C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2 for (B) ERK1 and (C) ERK2. Results are expressed as mean ± SEM ( n = 3).
Figure Legend Snippet: Time course of IFNγ-induced activation of p-ERK1/2 in BV-2 microglial cells. (A) Western blot analysis of a typical time course for IFNγ (10 ng/ml) to induce ERK1/2 phosphorylation in BV-2 microglia cells. Cell lysates were extracted at the time indicated. (B, C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2 for (B) ERK1 and (C) ERK2. Results are expressed as mean ± SEM ( n = 3).

Techniques Used: Activation Assay, Western Blot

Role of ERK1/2 activation in IFNγ-induced ROS and NO production in BV-2 and HAPI microglial cells. Cells were treated with IFNγ (10 ng/ml) with or without the MEK1/2 inhibitor, U0126 (1.25 to 10 μM). (A) Representative Western blot demonstrated ability for U0126 to inhibit phosphorylation of ERK1/2 dose-dependently 4 h after IFNγ treatment. (B) For ROS production, BV-2 cells were pretreated with different concentrations of U0126 for 1 h prior to stimulation with IFNγ for 12 h. (C) For NO production, both BV-2 and HAPI cells were pretreated with different concentrations of U0126 for 1 h prior to stimulation with IFNγ for 16 h. Results are expressed as the mean ± SEM ( n = 3), and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, ** P
Figure Legend Snippet: Role of ERK1/2 activation in IFNγ-induced ROS and NO production in BV-2 and HAPI microglial cells. Cells were treated with IFNγ (10 ng/ml) with or without the MEK1/2 inhibitor, U0126 (1.25 to 10 μM). (A) Representative Western blot demonstrated ability for U0126 to inhibit phosphorylation of ERK1/2 dose-dependently 4 h after IFNγ treatment. (B) For ROS production, BV-2 cells were pretreated with different concentrations of U0126 for 1 h prior to stimulation with IFNγ for 12 h. (C) For NO production, both BV-2 and HAPI cells were pretreated with different concentrations of U0126 for 1 h prior to stimulation with IFNγ for 16 h. Results are expressed as the mean ± SEM ( n = 3), and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, ** P

Techniques Used: Activation Assay, Western Blot

Hon and Mag inhibit IFNγ-induced activation of p-ERK1/2 in BV-2 microglial cells. (A) Western blot analysis showing a representative experiment of Hon or Mag pretreatment on IFNγ to induce p-ERK1/2 phosphorylation in BV-2 microglia cells. Cells were treated with either Hon or Mag (1 to 10 μM) for 1 h followed by stimulation with IFNγ (10 ng/ml) for 4 h. (B C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2. Results are expressed as the mean ± SEM ( n = 3) and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, * P
Figure Legend Snippet: Hon and Mag inhibit IFNγ-induced activation of p-ERK1/2 in BV-2 microglial cells. (A) Western blot analysis showing a representative experiment of Hon or Mag pretreatment on IFNγ to induce p-ERK1/2 phosphorylation in BV-2 microglia cells. Cells were treated with either Hon or Mag (1 to 10 μM) for 1 h followed by stimulation with IFNγ (10 ng/ml) for 4 h. (B C) Results of protein band intensities are expressed as arbitrary units of phospho-ERK1/2 against total ERK1/2. Results are expressed as the mean ± SEM ( n = 3) and significant difference from the respective IFNγ stimulated group was determined by one-way ANOVA followed by Dunnett’s tests, * P

Techniques Used: Activation Assay, Western Blot

35) Product Images from "Gravin orchestrates PKA and ?2-adrenergic receptor signaling critical for synaptic plasticity and memory"

Article Title: Gravin orchestrates PKA and ?2-adrenergic receptor signaling critical for synaptic plasticity and memory

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.3612-12.2012

Phosphorylation of the β2 adrenergic receptor and ERK1/2 is reduced in the hippocampus of gravin GT mice 30 minutes after fear conditioning
Figure Legend Snippet: Phosphorylation of the β2 adrenergic receptor and ERK1/2 is reduced in the hippocampus of gravin GT mice 30 minutes after fear conditioning

Techniques Used: Mouse Assay

36) Product Images from "PI3K/AKT AND ERK REGULATE RETINOIC ACID-INDUCED NEUROBLASTOMA CELLULAR DIFFERENTIATION"

Article Title: PI3K/AKT AND ERK REGULATE RETINOIC ACID-INDUCED NEUROBLASTOMA CELLULAR DIFFERENTIATION

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2012.06.125

RA phosphorylated AKT ERK1/2 and PI3K MAPK regulated RXR transcription activity
Figure Legend Snippet: RA phosphorylated AKT ERK1/2 and PI3K MAPK regulated RXR transcription activity

Techniques Used: Activity Assay

37) Product Images from "Membrane estrogen receptor-? levels predict estrogen-induced ERK1/2 activation in MCF-7 cells"

Article Title: Membrane estrogen receptor-? levels predict estrogen-induced ERK1/2 activation in MCF-7 cells

Journal: Breast Cancer Research

doi: 10.1186/bcr959

Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E 2 ), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mER high ) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E 2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E 2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.
Figure Legend Snippet: Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E 2 ), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mER high ) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E 2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E 2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.

Techniques Used: Activation Assay

Time course of extracellular signal-regulated kinase (ERK)1/2 activation with 17β-estradiol (E 2 ) and impeded ligand in membrane estrogen receptor (mER)-α-enriched (mER high ) and mER-α-depleted (mER low ) MCF-7 cells, and receptor-less cells. After 72 hours in medium with dextran-coated charcoal-stripped serum (DCSS), cells were treated with 1 pmol/l E 2 , or an equivalent concentration of hormone contained in an E 2 -peroxidase conjugate, for different time intervals. (a, b) mER high MCF-7 cells treated with E 2 and E 2 -peroxidase, respectively. (c) mER low MCF-7 cells treated with E 2 . (d) Estrogen receptor-α-negative MDA-MB-231 cells treated with E 2 . Each experiment was repeated at least three times, each time point represents 16 replicate wells, and the values are expressed as mean ± standard error. Insets: inhibition of ERK1/2 activation with U0126 upstream mitogen-activated protein kinase kinase (MEK)1/2 inhibitor. The cells were pretreated for 15 min with 40 μmol/l inhibitor (gray bars) and E 2 -induced activation of ERK1/2 was tested at activation times appropriate for each cell type (after 3, 6 and 10 min in the case of mER high or 6 min in the case of mER low cells). The asterisks indicate the significant reduction in ERK1/2 activation compared with cells treated only with E 2 (black bars).
Figure Legend Snippet: Time course of extracellular signal-regulated kinase (ERK)1/2 activation with 17β-estradiol (E 2 ) and impeded ligand in membrane estrogen receptor (mER)-α-enriched (mER high ) and mER-α-depleted (mER low ) MCF-7 cells, and receptor-less cells. After 72 hours in medium with dextran-coated charcoal-stripped serum (DCSS), cells were treated with 1 pmol/l E 2 , or an equivalent concentration of hormone contained in an E 2 -peroxidase conjugate, for different time intervals. (a, b) mER high MCF-7 cells treated with E 2 and E 2 -peroxidase, respectively. (c) mER low MCF-7 cells treated with E 2 . (d) Estrogen receptor-α-negative MDA-MB-231 cells treated with E 2 . Each experiment was repeated at least three times, each time point represents 16 replicate wells, and the values are expressed as mean ± standard error. Insets: inhibition of ERK1/2 activation with U0126 upstream mitogen-activated protein kinase kinase (MEK)1/2 inhibitor. The cells were pretreated for 15 min with 40 μmol/l inhibitor (gray bars) and E 2 -induced activation of ERK1/2 was tested at activation times appropriate for each cell type (after 3, 6 and 10 min in the case of mER high or 6 min in the case of mER low cells). The asterisks indicate the significant reduction in ERK1/2 activation compared with cells treated only with E 2 (black bars).

Techniques Used: Activation Assay, Concentration Assay, Multiple Displacement Amplification, Inhibition

Effects of phosphatase inhibitors on extracellular signal-regulated kinase (ERK)1/2 dephosphorylation. Cells were pretreated for 1 hour with different phosphatase inhibitors: 50 nmol/l okadaic acid (OA) or 3 μmol/l cyclosporin A (CyclA). Then, ERK1/2 phosphorylation was determined after 1 pmol/l E 2 stimulation for (a, b) 20 min or (c) 60 min. Black bars are membrane estrogen receptor (mER)-α-enriched (mER high ) MCF-7 cells; open bars are mER-α-depleted (mER low ) MCF-7 cells. The data are averages of two or three experiments, each with 24 replicates; values are expressed as mean ± standard error. The asterisks indicate significant differences from the vehicle control-treated samples with corresponding inhibitor included.
Figure Legend Snippet: Effects of phosphatase inhibitors on extracellular signal-regulated kinase (ERK)1/2 dephosphorylation. Cells were pretreated for 1 hour with different phosphatase inhibitors: 50 nmol/l okadaic acid (OA) or 3 μmol/l cyclosporin A (CyclA). Then, ERK1/2 phosphorylation was determined after 1 pmol/l E 2 stimulation for (a, b) 20 min or (c) 60 min. Black bars are membrane estrogen receptor (mER)-α-enriched (mER high ) MCF-7 cells; open bars are mER-α-depleted (mER low ) MCF-7 cells. The data are averages of two or three experiments, each with 24 replicates; values are expressed as mean ± standard error. The asterisks indicate significant differences from the vehicle control-treated samples with corresponding inhibitor included.

Techniques Used: De-Phosphorylation Assay

38) Product Images from "Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration"

Article Title: Bilirubin Inhibits Neointima Formation and Vascular Smooth Muscle Cell Proliferation and Migration

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2012.00048

Effect of bilirubin on the expression or phosphorylation of cell cycle regulatory proteins, ERK1/2, or HO-1 following rat carotid artery balloon injury . (A) Expression of cyclin D1, cyclin E, cyclin A, p21, p27, p53, and β-actin protein in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. (B) Expression of phospho-ERK1/2 and total ERK1/2 in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. (C) Expression of HO-1 in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. Quantification of relative protein levels was achieved by scanning densitometry. Results are means ± SEM ( n = 3–5). *Statistically significant effect of arterial injury. + Statistically significant effect of bilirubin.
Figure Legend Snippet: Effect of bilirubin on the expression or phosphorylation of cell cycle regulatory proteins, ERK1/2, or HO-1 following rat carotid artery balloon injury . (A) Expression of cyclin D1, cyclin E, cyclin A, p21, p27, p53, and β-actin protein in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. (B) Expression of phospho-ERK1/2 and total ERK1/2 in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. (C) Expression of HO-1 in control and injured arteries treated with an empty (E) gel or a gel containing bilirubin (B) 2 days after injury. Quantification of relative protein levels was achieved by scanning densitometry. Results are means ± SEM ( n = 3–5). *Statistically significant effect of arterial injury. + Statistically significant effect of bilirubin.

Techniques Used: Expressing

39) Product Images from "Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells"

Article Title: Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells

Journal: International Journal of Oncology

doi: 10.3892/ijo.2012.1635

Downregulation of protein kinase CK2 affects the mTOR and ERK1/2 signaling pathways. Cell lysates (30 μ g) from cells treated as indicated in the figure were analyzed by western blot analysis using antibodies against the indicated proteins or their phosphorylated form. Anti-β-actin was applied to confirm equal protein loading. At least 4 separate experiments were performed obtaining similar results. Data from one representative experiment are shown.
Figure Legend Snippet: Downregulation of protein kinase CK2 affects the mTOR and ERK1/2 signaling pathways. Cell lysates (30 μ g) from cells treated as indicated in the figure were analyzed by western blot analysis using antibodies against the indicated proteins or their phosphorylated form. Anti-β-actin was applied to confirm equal protein loading. At least 4 separate experiments were performed obtaining similar results. Data from one representative experiment are shown.

Techniques Used: Western Blot

CK2-knockout leads to enhanced ERK1/2 kinase activity in the presence of NCS. Cells were transfected with Scr-siRNA or CK2-siRNAs for 72 h. Where indicated, NCS was added 24 h before harvesting. Whole cell lysates (500 μ g) were subjected to a non-radioactive kinase activity assay in the presence of a GST-Elk-1 fusion protein after immunoprecipitation of phosphorylated ERK1/2. The phosphorylation levels of Elk-1 were detected by western blot analysis with an antibody directed against phospho-Elk1-1 (S383). Representative results from two independent experiments are shown.
Figure Legend Snippet: CK2-knockout leads to enhanced ERK1/2 kinase activity in the presence of NCS. Cells were transfected with Scr-siRNA or CK2-siRNAs for 72 h. Where indicated, NCS was added 24 h before harvesting. Whole cell lysates (500 μ g) were subjected to a non-radioactive kinase activity assay in the presence of a GST-Elk-1 fusion protein after immunoprecipitation of phosphorylated ERK1/2. The phosphorylation levels of Elk-1 were detected by western blot analysis with an antibody directed against phospho-Elk1-1 (S383). Representative results from two independent experiments are shown.

Techniques Used: Knock-Out, Activity Assay, Transfection, Kinase Assay, Immunoprecipitation, Western Blot

Model of the proposed influence of CK2 on autophagy induction. The model suggests the placement and function of CK2 with respect to the major signaling cascades regulating autophagy. CK2 has been reported to be a master regulator of cellular functions in virtue of its ability to play a ‘lateral means’ of pathways intervention ( 12 ). Here, we show that effective CK2-mediated autophagy induction is achieved by the simultaneous targeting of the ERK1/2-and mTOR signaling pathways. Additional details are reported in the text.
Figure Legend Snippet: Model of the proposed influence of CK2 on autophagy induction. The model suggests the placement and function of CK2 with respect to the major signaling cascades regulating autophagy. CK2 has been reported to be a master regulator of cellular functions in virtue of its ability to play a ‘lateral means’ of pathways intervention ( 12 ). Here, we show that effective CK2-mediated autophagy induction is achieved by the simultaneous targeting of the ERK1/2-and mTOR signaling pathways. Additional details are reported in the text.

Techniques Used:

40) Product Images from "Membrane estrogen receptor-? levels predict estrogen-induced ERK1/2 activation in MCF-7 cells"

Article Title: Membrane estrogen receptor-? levels predict estrogen-induced ERK1/2 activation in MCF-7 cells

Journal: Breast Cancer Research

doi: 10.1186/bcr959

Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E 2 ), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mER high ) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E 2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E 2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.
Figure Legend Snippet: Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E 2 ), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mER high ) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E 2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E 2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.

Techniques Used: Activation Assay

Time course of extracellular signal-regulated kinase (ERK)1/2 activation with 17β-estradiol (E 2 ) and impeded ligand in membrane estrogen receptor (mER)-α-enriched (mER high ) and mER-α-depleted (mER low ) MCF-7 cells, and receptor-less cells. After 72 hours in medium with dextran-coated charcoal-stripped serum (DCSS), cells were treated with 1 pmol/l E 2 , or an equivalent concentration of hormone contained in an E 2 -peroxidase conjugate, for different time intervals. (a, b) mER high MCF-7 cells treated with E 2 and E 2 -peroxidase, respectively. (c) mER low MCF-7 cells treated with E 2 . (d) Estrogen receptor-α-negative MDA-MB-231 cells treated with E 2 . Each experiment was repeated at least three times, each time point represents 16 replicate wells, and the values are expressed as mean ± standard error. Insets: inhibition of ERK1/2 activation with U0126 upstream mitogen-activated protein kinase kinase (MEK)1/2 inhibitor. The cells were pretreated for 15 min with 40 μmol/l inhibitor (gray bars) and E 2 -induced activation of ERK1/2 was tested at activation times appropriate for each cell type (after 3, 6 and 10 min in the case of mER high or 6 min in the case of mER low cells). The asterisks indicate the significant reduction in ERK1/2 activation compared with cells treated only with E 2 (black bars).
Figure Legend Snippet: Time course of extracellular signal-regulated kinase (ERK)1/2 activation with 17β-estradiol (E 2 ) and impeded ligand in membrane estrogen receptor (mER)-α-enriched (mER high ) and mER-α-depleted (mER low ) MCF-7 cells, and receptor-less cells. After 72 hours in medium with dextran-coated charcoal-stripped serum (DCSS), cells were treated with 1 pmol/l E 2 , or an equivalent concentration of hormone contained in an E 2 -peroxidase conjugate, for different time intervals. (a, b) mER high MCF-7 cells treated with E 2 and E 2 -peroxidase, respectively. (c) mER low MCF-7 cells treated with E 2 . (d) Estrogen receptor-α-negative MDA-MB-231 cells treated with E 2 . Each experiment was repeated at least three times, each time point represents 16 replicate wells, and the values are expressed as mean ± standard error. Insets: inhibition of ERK1/2 activation with U0126 upstream mitogen-activated protein kinase kinase (MEK)1/2 inhibitor. The cells were pretreated for 15 min with 40 μmol/l inhibitor (gray bars) and E 2 -induced activation of ERK1/2 was tested at activation times appropriate for each cell type (after 3, 6 and 10 min in the case of mER high or 6 min in the case of mER low cells). The asterisks indicate the significant reduction in ERK1/2 activation compared with cells treated only with E 2 (black bars).

Techniques Used: Activation Assay, Concentration Assay, Multiple Displacement Amplification, Inhibition

Effects of phosphatase inhibitors on extracellular signal-regulated kinase (ERK)1/2 dephosphorylation. Cells were pretreated for 1 hour with different phosphatase inhibitors: 50 nmol/l okadaic acid (OA) or 3 μmol/l cyclosporin A (CyclA). Then, ERK1/2 phosphorylation was determined after 1 pmol/l E 2 stimulation for (a, b) 20 min or (c) 60 min. Black bars are membrane estrogen receptor (mER)-α-enriched (mER high ) MCF-7 cells; open bars are mER-α-depleted (mER low ) MCF-7 cells. The data are averages of two or three experiments, each with 24 replicates; values are expressed as mean ± standard error. The asterisks indicate significant differences from the vehicle control-treated samples with corresponding inhibitor included.
Figure Legend Snippet: Effects of phosphatase inhibitors on extracellular signal-regulated kinase (ERK)1/2 dephosphorylation. Cells were pretreated for 1 hour with different phosphatase inhibitors: 50 nmol/l okadaic acid (OA) or 3 μmol/l cyclosporin A (CyclA). Then, ERK1/2 phosphorylation was determined after 1 pmol/l E 2 stimulation for (a, b) 20 min or (c) 60 min. Black bars are membrane estrogen receptor (mER)-α-enriched (mER high ) MCF-7 cells; open bars are mER-α-depleted (mER low ) MCF-7 cells. The data are averages of two or three experiments, each with 24 replicates; values are expressed as mean ± standard error. The asterisks indicate significant differences from the vehicle control-treated samples with corresponding inhibitor included.

Techniques Used: De-Phosphorylation Assay

41) Product Images from "Caveolin-1 Orchestrates Fibroblast Growth Factor 2 Signaling Control of Angiogenesis in Placental Artery Endothelial Cell Caveolae"

Article Title: Caveolin-1 Orchestrates Fibroblast Growth Factor 2 Signaling Control of Angiogenesis in Placental Artery Endothelial Cell Caveolae

Journal: Journal of Cellular Physiology

doi: 10.1002/jcp.22984

FGF2 stimulates oFPAE cell proliferation, migration and tube formation – role of PI3K/AKT1 and ERK1/2
Figure Legend Snippet: FGF2 stimulates oFPAE cell proliferation, migration and tube formation – role of PI3K/AKT1 and ERK1/2

Techniques Used: Migration

FGF2 activates AKT1 and ERK1/2 in oFPAE cell caveolae
Figure Legend Snippet: FGF2 activates AKT1 and ERK1/2 in oFPAE cell caveolae

Techniques Used:

Role of caveolin-1 in FGF2-induced activation of AKT1 and ERK1/2 in oFPAE cells
Figure Legend Snippet: Role of caveolin-1 in FGF2-induced activation of AKT1 and ERK1/2 in oFPAE cells

Techniques Used: Activation Assay

42) Product Images from "Upregulation of SIRT6 predicts poor prognosis and promotes metastasis of non-small cell lung cancer via the ERK1/2/MMP9 pathway"

Article Title: Upregulation of SIRT6 predicts poor prognosis and promotes metastasis of non-small cell lung cancer via the ERK1/2/MMP9 pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.9750

SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.
Figure Legend Snippet: SIRT6 promotes NSCLC cell migration and invasion through ERK1/2//MMP9 Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing NSCLC cells and corresponding vector control cells ( A ) Western blotting analysis of p-ERK1/2, ERK1/2 and MMP9 in indicated cells ( B and D ) SIRT6 overexpression increased p-ERK1/2 and MMP9 expression, and treatment with the specific MEK1/2 inhibitor U0126 abolishes these effects. Zymographic analysis of MMP9 activity in conditioned medium from SIRT6-overexpressing cells, with or without U0126 ( C ) Wound healing assay results showed that stable SIRT6 overexpression promotes cell migration, which is abolished by concomitant treatment with U0126 ( E ) Migration and invasion assays using a transwell assay system ( F ) SIRT6-overexpressing cells were treated with U0126 or vehicle alone (without U0126). Representative images and quantification of migration and invasion are shown.

Techniques Used: Migration, Activity Assay, Plasmid Preparation, Western Blot, Over Expression, Expressing, Wound Healing Assay, Transwell Assay

43) Product Images from "Catalpol Protects Pre-Myelinating Oligodendrocytes against Ischemia-induced Oxidative Injury through ERK1/2 Signaling Pathway"

Article Title: Catalpol Protects Pre-Myelinating Oligodendrocytes against Ischemia-induced Oxidative Injury through ERK1/2 Signaling Pathway

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.16823

Effects of blocking ERK1/2 pathway on the oxidative injury of PreOLs under OGD. (A-D) Representative TUNEL staining (red) patterns in the CTL (A), OGD (B), U0126 (C), and U0126+CAT (D) groups. Cell nuclei were counterstained with DAPI (blue). (E) Quantification of TUNEL-positive cells in the CTL, OGD, U0126, and U0126+CAT groups. (F) Quantification of ROS levels in the CTL, OGD, U0126, and U0126+CAT groups. (G) Quantification of red/green fluorescence ratios after JC-1 staining in the CTL, OGD, U0126, and U0126+CAT groups. (H) Quantification of the average fluorescence intensities of Fluo-3 in the CTL, OGD, U0126, and U0126+CAT groups. Data are expressed as percentages of the values in the CTL group and are shown as the means ± SEM (n = 6 in each group). * p
Figure Legend Snippet: Effects of blocking ERK1/2 pathway on the oxidative injury of PreOLs under OGD. (A-D) Representative TUNEL staining (red) patterns in the CTL (A), OGD (B), U0126 (C), and U0126+CAT (D) groups. Cell nuclei were counterstained with DAPI (blue). (E) Quantification of TUNEL-positive cells in the CTL, OGD, U0126, and U0126+CAT groups. (F) Quantification of ROS levels in the CTL, OGD, U0126, and U0126+CAT groups. (G) Quantification of red/green fluorescence ratios after JC-1 staining in the CTL, OGD, U0126, and U0126+CAT groups. (H) Quantification of the average fluorescence intensities of Fluo-3 in the CTL, OGD, U0126, and U0126+CAT groups. Data are expressed as percentages of the values in the CTL group and are shown as the means ± SEM (n = 6 in each group). * p

Techniques Used: Blocking Assay, TUNEL Assay, Staining, CTL Assay, Fluorescence

Effects of catalpol on the activation of the ERK1/2 pathway in PreOLs under OGD. (A) Western blot analysis for p-ERK1/2 and PARP-1 expression in the CTL, OGD, CAT, and U0126 groups. Total ERK1/2 and GAPDH were used as loading controls. The blot shown is representative of six independent experiments. (B) Quantification of p-ERK1/2 and PARP-1 protein expression levels from Western blot analysis. Data are expressed as percentages of the values in the CTL group and are shown as the means ± SEM (n = 6 in each group). * p
Figure Legend Snippet: Effects of catalpol on the activation of the ERK1/2 pathway in PreOLs under OGD. (A) Western blot analysis for p-ERK1/2 and PARP-1 expression in the CTL, OGD, CAT, and U0126 groups. Total ERK1/2 and GAPDH were used as loading controls. The blot shown is representative of six independent experiments. (B) Quantification of p-ERK1/2 and PARP-1 protein expression levels from Western blot analysis. Data are expressed as percentages of the values in the CTL group and are shown as the means ± SEM (n = 6 in each group). * p

Techniques Used: Activation Assay, Western Blot, Expressing, CTL Assay

44) Product Images from "CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells"

Article Title: CXCL7-Mediated Stimulation of Lymphangiogenic Factors VEGF-C, VEGF-D in Human Breast Cancer Cells

Journal: Journal of Oncology

doi: 10.1155/2010/939407

ERK1/2 mitogen-activated protein kinase is not active in CXCL7 stable transfected MCF10AT cells. Briefly, the cells were harvested in PBS, counted and lysed in the RIPA buffer with protease inhibitor cocktail. Protein concentration was determined for all samples using the Bio-Rad protein assay. The equal-volume samples (50 μ g) were separated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane. Immunodetection was performed using pERK1/2 and ERK1/2, then developed by ECL. Stable CXCL7 transfected MCF10AT cells did not induce ERK1/2 phosphorylation compared to the vector transfected control cells. Furthermore, the expression of ERK1/2 was the same in stable CXCL7 transfected MCF10AT cells compared to vector controls.
Figure Legend Snippet: ERK1/2 mitogen-activated protein kinase is not active in CXCL7 stable transfected MCF10AT cells. Briefly, the cells were harvested in PBS, counted and lysed in the RIPA buffer with protease inhibitor cocktail. Protein concentration was determined for all samples using the Bio-Rad protein assay. The equal-volume samples (50 μ g) were separated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane. Immunodetection was performed using pERK1/2 and ERK1/2, then developed by ECL. Stable CXCL7 transfected MCF10AT cells did not induce ERK1/2 phosphorylation compared to the vector transfected control cells. Furthermore, the expression of ERK1/2 was the same in stable CXCL7 transfected MCF10AT cells compared to vector controls.

Techniques Used: Transfection, Protease Inhibitor, Protein Concentration, SDS Page, Immunodetection, Plasmid Preparation, Expressing

45) Product Images from "Spinal Adenosine A2a Receptor Activation Elicits Long-Lasting Phrenic Motor Facilitation"

Article Title: Spinal Adenosine A2a Receptor Activation Elicits Long-Lasting Phrenic Motor Facilitation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3570-07.2008

A2a-induced facilitation of phrenic motor output requires tyrosine kinase activation, and C4 A2a receptor activation phosphorylates immature TrkB 80 protein near phrenic motoneurons. a , Average data demonstrating that intrathecal K252a (1.75 μg · kg −1 ; squares; n = 5), a tyrosine kinase inhibitor, blocked A2a-induced phrenic motor facilitation (circles; n = 12), but only 60 min after intrathecal CGS 21680 injection. b , Representative immunoblot for TrkB and phospho-TrkB (pTrkB) protein, and Akt and phospho-Akt protein from C4 ventral spinal cords collected from two rats at 120 min after intrathecal vehicle (DMSO) or CGS 21680 injections. c , Average data depicting percentage change from vehicle of C4 ventral spinal TrkB (normalized to GAPDH) and pTrkB (normalized to total TrkB) protein isoforms after intrathecal vehicle ( n = 6) or CGS 21680 (CGS; n = 9) injection. Intrathecal CGS 21680 increased mature (150 kDa) and immature (∼80 kDa) isoforms of TrkB protein. Intrathecal CGS 21680 also selectively phosphorylated the low-molecular-weight TrkB isoform. d , Average data depicting percentage change from vehicle ( n = 6) of C4 ventral spinal Akt, ERK1, and ERK2 after intrathecal vehicle or CGS 21680 ( n = 9) injection. Intrathecal CGS 21680 activated Akt but not ERK1/2 or PLC-γ. e , Immunoblot from the peptide blocking experiment. TrkB antibody was preincubated with TrkB-blocking protein to decrease TrkB antibody binding to TrkB receptor protein on the C4 ventral spinal immunoblots. f , Immunoblot from the deglycosylation experiment. PNGase was added to C4 ventral spinal protein. The intensity of the ∼80 kDa TrkB isoform decreased, and the ∼50 kDa core protein intensity increased, after the digestion. *Significantly different from other groups ( p
Figure Legend Snippet: A2a-induced facilitation of phrenic motor output requires tyrosine kinase activation, and C4 A2a receptor activation phosphorylates immature TrkB 80 protein near phrenic motoneurons. a , Average data demonstrating that intrathecal K252a (1.75 μg · kg −1 ; squares; n = 5), a tyrosine kinase inhibitor, blocked A2a-induced phrenic motor facilitation (circles; n = 12), but only 60 min after intrathecal CGS 21680 injection. b , Representative immunoblot for TrkB and phospho-TrkB (pTrkB) protein, and Akt and phospho-Akt protein from C4 ventral spinal cords collected from two rats at 120 min after intrathecal vehicle (DMSO) or CGS 21680 injections. c , Average data depicting percentage change from vehicle of C4 ventral spinal TrkB (normalized to GAPDH) and pTrkB (normalized to total TrkB) protein isoforms after intrathecal vehicle ( n = 6) or CGS 21680 (CGS; n = 9) injection. Intrathecal CGS 21680 increased mature (150 kDa) and immature (∼80 kDa) isoforms of TrkB protein. Intrathecal CGS 21680 also selectively phosphorylated the low-molecular-weight TrkB isoform. d , Average data depicting percentage change from vehicle ( n = 6) of C4 ventral spinal Akt, ERK1, and ERK2 after intrathecal vehicle or CGS 21680 ( n = 9) injection. Intrathecal CGS 21680 activated Akt but not ERK1/2 or PLC-γ. e , Immunoblot from the peptide blocking experiment. TrkB antibody was preincubated with TrkB-blocking protein to decrease TrkB antibody binding to TrkB receptor protein on the C4 ventral spinal immunoblots. f , Immunoblot from the deglycosylation experiment. PNGase was added to C4 ventral spinal protein. The intensity of the ∼80 kDa TrkB isoform decreased, and the ∼50 kDa core protein intensity increased, after the digestion. *Significantly different from other groups ( p

Techniques Used: Activation Assay, Injection, Molecular Weight, Planar Chromatography, Blocking Assay, Binding Assay, Western Blot

46) Product Images from "Activation of PI3K Is Indispensable for Interleukin 7-mediated Viability, Proliferation, Glucose Use, and Growth of T Cell Acute Lymphoblastic Leukemia Cells"

Article Title: Activation of PI3K Is Indispensable for Interleukin 7-mediated Viability, Proliferation, Glucose Use, and Growth of T Cell Acute Lymphoblastic Leukemia Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20040789

IL-7 induces PI3K-dependent phosphorylation of Akt, GSK-3, FOXO1, and FOXO3a, and MEK-dependent phosphorylation of Erk1/2 in T-ALL cells. IL-7–deprived TAIL7 cells were pretreated with 10 μM LY294002 (LY) or 10 μM PD098059 (PD) for 2 h, and then stimulated with IL-7 for 15 min. (A) Western blot analysis was performed with P-Erk1/2, P-Akt antibodies (see legend to Fig. 1 ), and an antibody specific for Tyr694/Tyr699-phosphorylated-STAT5A/B (P-STAT5) to confirm that LY294002 and PD98059 were specific inhibitors of the PI3K–Akt and MEK–Erk pathway, respectively. (B) GSK-3β, FOXO1, and FOXO3a phosphorylation is dependent on PI3K activity. Western blot analysis was performed with P-Akt, P-GSK3β, and P-FOXO1/FOXO3a antibodies. Anti-STAT5 (A) and actin (B) antibodies were used to confirm equal loading. Representative results from three independent experiments are shown.
Figure Legend Snippet: IL-7 induces PI3K-dependent phosphorylation of Akt, GSK-3, FOXO1, and FOXO3a, and MEK-dependent phosphorylation of Erk1/2 in T-ALL cells. IL-7–deprived TAIL7 cells were pretreated with 10 μM LY294002 (LY) or 10 μM PD098059 (PD) for 2 h, and then stimulated with IL-7 for 15 min. (A) Western blot analysis was performed with P-Erk1/2, P-Akt antibodies (see legend to Fig. 1 ), and an antibody specific for Tyr694/Tyr699-phosphorylated-STAT5A/B (P-STAT5) to confirm that LY294002 and PD98059 were specific inhibitors of the PI3K–Akt and MEK–Erk pathway, respectively. (B) GSK-3β, FOXO1, and FOXO3a phosphorylation is dependent on PI3K activity. Western blot analysis was performed with P-Akt, P-GSK3β, and P-FOXO1/FOXO3a antibodies. Anti-STAT5 (A) and actin (B) antibodies were used to confirm equal loading. Representative results from three independent experiments are shown.

Techniques Used: Western Blot, Activity Assay

IL-7 activates MEK–Erk and PI3K–Akt pathways in T-ALL. IL-7–deprived TAIL7 cells were stimulated with IL-7 for the indicated periods (A and B). Cell lysates were resolved with 10% SDS-PAGE and immunoblotted with the indicated antibodies. Results representative of three independent experiments are shown. Levels of phosphorylated MEK1/2 and Erk1/2 were detected with antisera that selectively recognize the activated forms of the following kinases: Ser217/Ser221-phosphorylated MEK1/2 (P-MEK1/2) and Thr202/Tyr204-dual-phosphorylated Erk1/2 (P-Erk1/2). Levels of phosphorylated Akt and GSK-3 were analyzed with antibodies that specifically recognize Ser473-phosphorylated Akt (P-Akt) and Ser9-phosphorylated GSK-3β (P-GSK-3β), respectively. Levels of phosphorylated FOXO1/FOXO3a were detected with antiserum that reacts with Thr24-phosphorylated FOXO1 (P-AFOXO1) and Thr32-phosphorylated FOXO3a (P- FOXO3a). Akt and Erk1/2 protein levels were assessed with specific antibodies and remained unchanged (not depicted). Blots were reprobed with an anti–ZAP-70 antibody to confirm even protein loading. (C) IL-7 activates Akt and induces in vitro phosphorylation of GSK-3 by Akt. IL-7–deprived TAIL7 cells were stimulated with IL-7 for 15 min. To compare Akt enzymatic activity in unstimulated (Unst.) versus IL-7–stimulated cells (IL-7), cell lysates were immunoprecipitated with agarose-conjugated anti-Akt antibody and in vitro kinase reactions were performed using crosstide-GSK-3α/β as exogenous substrate. Reactions were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membrane, and GSK-3 phosphorylation was detected by immunoblotting with anti–phospho-GSK-3α/β (Ser21/Ser9) antibody. Even loading was confirmed with an anti-Akt antibody. Relative quantification of phosphorylated GSK-3α and GSK-3β bands was performed by densitometry analysis. Results were normalized in relation to the loading control (Akt) and expressed as relative units. IL-7 induced a 1.63-fold increase in GSK-3α and a 3.07-fold increase in GSK-3β Akt-mediated phosphorylation. Results are representative of two independent experiments.
Figure Legend Snippet: IL-7 activates MEK–Erk and PI3K–Akt pathways in T-ALL. IL-7–deprived TAIL7 cells were stimulated with IL-7 for the indicated periods (A and B). Cell lysates were resolved with 10% SDS-PAGE and immunoblotted with the indicated antibodies. Results representative of three independent experiments are shown. Levels of phosphorylated MEK1/2 and Erk1/2 were detected with antisera that selectively recognize the activated forms of the following kinases: Ser217/Ser221-phosphorylated MEK1/2 (P-MEK1/2) and Thr202/Tyr204-dual-phosphorylated Erk1/2 (P-Erk1/2). Levels of phosphorylated Akt and GSK-3 were analyzed with antibodies that specifically recognize Ser473-phosphorylated Akt (P-Akt) and Ser9-phosphorylated GSK-3β (P-GSK-3β), respectively. Levels of phosphorylated FOXO1/FOXO3a were detected with antiserum that reacts with Thr24-phosphorylated FOXO1 (P-AFOXO1) and Thr32-phosphorylated FOXO3a (P- FOXO3a). Akt and Erk1/2 protein levels were assessed with specific antibodies and remained unchanged (not depicted). Blots were reprobed with an anti–ZAP-70 antibody to confirm even protein loading. (C) IL-7 activates Akt and induces in vitro phosphorylation of GSK-3 by Akt. IL-7–deprived TAIL7 cells were stimulated with IL-7 for 15 min. To compare Akt enzymatic activity in unstimulated (Unst.) versus IL-7–stimulated cells (IL-7), cell lysates were immunoprecipitated with agarose-conjugated anti-Akt antibody and in vitro kinase reactions were performed using crosstide-GSK-3α/β as exogenous substrate. Reactions were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membrane, and GSK-3 phosphorylation was detected by immunoblotting with anti–phospho-GSK-3α/β (Ser21/Ser9) antibody. Even loading was confirmed with an anti-Akt antibody. Relative quantification of phosphorylated GSK-3α and GSK-3β bands was performed by densitometry analysis. Results were normalized in relation to the loading control (Akt) and expressed as relative units. IL-7 induced a 1.63-fold increase in GSK-3α and a 3.07-fold increase in GSK-3β Akt-mediated phosphorylation. Results are representative of two independent experiments.

Techniques Used: SDS Page, In Vitro, Activity Assay, Immunoprecipitation

47) Product Images from "PVT1 dependence in cancer with MYC copy-number increase"

Article Title: PVT1 dependence in cancer with MYC copy-number increase

Journal: Nature

doi: 10.1038/nature13311

Abnormal oncogenic stress, proliferation and differentiation in gain(Myc,Pvt1,Ccdc26, Gsdmc) mammary ducts a , b , Western blot analysis of p53 ( a ) and phospho-Erk1/2 ( b ) in total protein lysates from mammary glands of indicated genotypes. The relative densities of p53 and p-ERK1/2 were calculated by normalizing against the GAPDH and total ERK1/2 protein levels respectively. c , Immunofluorescence analysis of ERα (green) on sections of mammary ducts. Cell nuclei positive for ERα are presented as the percentage of total epithelial cell nuclei (DAPI, blue). Images shown are representative three mice per genotype. d , Haematoxylin and eosin staining of the mammary ducts from wild type and gain (M,P,C,G) mice showed precocious alveolar-like phenotype in the latter. This aberrant structure is shown at higher magnification in the right row. e , Immunofluorescence co-staining for DAPI (blue), luminal marker K8 (red) and myoepithelial marker K14 (green) in mice. Arrowheads indicate co-expression of K8 and K14. DAPI-stained nuclei in blue. Mean ± s.e.m. for a–c ( n = 3). * P
Figure Legend Snippet: Abnormal oncogenic stress, proliferation and differentiation in gain(Myc,Pvt1,Ccdc26, Gsdmc) mammary ducts a , b , Western blot analysis of p53 ( a ) and phospho-Erk1/2 ( b ) in total protein lysates from mammary glands of indicated genotypes. The relative densities of p53 and p-ERK1/2 were calculated by normalizing against the GAPDH and total ERK1/2 protein levels respectively. c , Immunofluorescence analysis of ERα (green) on sections of mammary ducts. Cell nuclei positive for ERα are presented as the percentage of total epithelial cell nuclei (DAPI, blue). Images shown are representative three mice per genotype. d , Haematoxylin and eosin staining of the mammary ducts from wild type and gain (M,P,C,G) mice showed precocious alveolar-like phenotype in the latter. This aberrant structure is shown at higher magnification in the right row. e , Immunofluorescence co-staining for DAPI (blue), luminal marker K8 (red) and myoepithelial marker K14 (green) in mice. Arrowheads indicate co-expression of K8 and K14. DAPI-stained nuclei in blue. Mean ± s.e.m. for a–c ( n = 3). * P

Techniques Used: Western Blot, Immunofluorescence, Mouse Assay, Staining, Marker, Expressing

48) Product Images from "Opiate Exposure and Withdrawal Induces a Molecular Memory Switch in the Basolateral Amygdala between ERK1/2 and CaMKIIα-Dependent Signaling Substrates"

Article Title: Opiate Exposure and Withdrawal Induces a Molecular Memory Switch in the Basolateral Amygdala between ERK1/2 and CaMKIIα-Dependent Signaling Substrates

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1226-13.2013

Effects of chronic opiate exposure and withdrawal on ERK1/2 and CaMKIIα signaling within the amygdala. A , Representative Western blot comparing total ERK1 and ERK2 levels across opiate-naive versus dependent/withdrawn experimental groups. B , Representative Western blot comparing pERK1 and pERK2 levels across opiate-exposure groups. C , Representative Western blot demonstrating dramatic downregulation of both total and phosphorylated levels of CaMKIIα across opiate-exposure groups. D , Representative Western blot demonstrating levels of total intra-BLA CaMKIIβ in either opiate-naive or opiate-dependent/withdrawn rats. E , Total levels of intra-BLA CaMKIIβ do not show significant differences between experimental opiate-exposure states.
Figure Legend Snippet: Effects of chronic opiate exposure and withdrawal on ERK1/2 and CaMKIIα signaling within the amygdala. A , Representative Western blot comparing total ERK1 and ERK2 levels across opiate-naive versus dependent/withdrawn experimental groups. B , Representative Western blot comparing pERK1 and pERK2 levels across opiate-exposure groups. C , Representative Western blot demonstrating dramatic downregulation of both total and phosphorylated levels of CaMKIIα across opiate-exposure groups. D , Representative Western blot demonstrating levels of total intra-BLA CaMKIIβ in either opiate-naive or opiate-dependent/withdrawn rats. E , Total levels of intra-BLA CaMKIIβ do not show significant differences between experimental opiate-exposure states.

Techniques Used: Western Blot

Effects of chronic opiate exposure and withdrawal on total and phosphorylated ERK1/2 protein expression levels in amygdala. A , Western blot analysis of total amygdalar levels of ERK1 and ERK2 expression revealed no difference in total ERK1 or ERK2 levels in either opiate-naive or opiate-dependent/withdrawn samples. B , In contrast, total pERK1 and pERK2 levels were significantly downregulated across naive versus dependent/withdrawn samples. C , D , Comparing pERK1/2 to total ERK1/2 ratios reveals a significant downregulation in relative pERK1/2 versus total ERK1/2 levels.
Figure Legend Snippet: Effects of chronic opiate exposure and withdrawal on total and phosphorylated ERK1/2 protein expression levels in amygdala. A , Western blot analysis of total amygdalar levels of ERK1 and ERK2 expression revealed no difference in total ERK1 or ERK2 levels in either opiate-naive or opiate-dependent/withdrawn samples. B , In contrast, total pERK1 and pERK2 levels were significantly downregulated across naive versus dependent/withdrawn samples. C , D , Comparing pERK1/2 to total ERK1/2 ratios reveals a significant downregulation in relative pERK1/2 versus total ERK1/2 levels.

Techniques Used: Expressing, Western Blot

49) Product Images from "Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation"

Article Title: Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302874

CD151 deficiency leads to enhanced and sustained ERK1/2 and Akt activation in IgE-stimulated mast cells. BMMCs from WT and CD151 −/− mice were sensitized with 1 μg/ml IgE for 24 h and then stimulated with 0.5 μg/ml DNP-HSA for the time points indicated. Cell lysates were subjected to immunoblotting analysis of the following phosphorylation events: Syk phosphorylation at Tyr 519/520 and Tyr 317 , PLCγ1 at Tyr 783 , Akt phosphorylation at Ser 473 , ERK1/2 phosphorylation at Thr 202 /Tyr 204 , and total phosphotyrosine detection, which represents total phosphorylation events following IgE stimulation. Representative Western blots are shown on the left , as phosphorylation bands followed by total protein loading controls. Bar graphs on the right show intensities of Western blot bands quantified by densitometry analysis. Fold increase in phosphorylation intensity was measured relative to total levels of detected proteins of interest. Densitometry values are mean ± SEM of three independent Western blots. Filled columns indicate WT; open columns indicate CD151 −/− . * p
Figure Legend Snippet: CD151 deficiency leads to enhanced and sustained ERK1/2 and Akt activation in IgE-stimulated mast cells. BMMCs from WT and CD151 −/− mice were sensitized with 1 μg/ml IgE for 24 h and then stimulated with 0.5 μg/ml DNP-HSA for the time points indicated. Cell lysates were subjected to immunoblotting analysis of the following phosphorylation events: Syk phosphorylation at Tyr 519/520 and Tyr 317 , PLCγ1 at Tyr 783 , Akt phosphorylation at Ser 473 , ERK1/2 phosphorylation at Thr 202 /Tyr 204 , and total phosphotyrosine detection, which represents total phosphorylation events following IgE stimulation. Representative Western blots are shown on the left , as phosphorylation bands followed by total protein loading controls. Bar graphs on the right show intensities of Western blot bands quantified by densitometry analysis. Fold increase in phosphorylation intensity was measured relative to total levels of detected proteins of interest. Densitometry values are mean ± SEM of three independent Western blots. Filled columns indicate WT; open columns indicate CD151 −/− . * p

Techniques Used: Activation Assay, Mouse Assay, Western Blot

50) Product Images from "Levodopa/Benserazide Loaded Microspheres Alleviate L-dopa Induced Dyskinesia through Preventing the Over-Expression of D1R/Shp-2/ERK1/2 Signaling Pathway in a Rat Model of Parkinson's Disease"

Article Title: Levodopa/Benserazide Loaded Microspheres Alleviate L-dopa Induced Dyskinesia through Preventing the Over-Expression of D1R/Shp-2/ERK1/2 Signaling Pathway in a Rat Model of Parkinson's Disease

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2017.00331

Changes in expression of phospho-ERK1/2, ERK1/2 after either intermittent or continuous L-dopa administration in 6-OHDA-lesioned striatum for 21 days. The expression of phospho-ERK1/2 in the LS group significantly increased compared with the sham, LBM-L and LBM-H group ( p
Figure Legend Snippet: Changes in expression of phospho-ERK1/2, ERK1/2 after either intermittent or continuous L-dopa administration in 6-OHDA-lesioned striatum for 21 days. The expression of phospho-ERK1/2 in the LS group significantly increased compared with the sham, LBM-L and LBM-H group ( p

Techniques Used: Expressing

51) Product Images from "Ketogenic Diet Impairs FGF21 Signaling and Promotes Differential Inflammatory Responses in the Liver and White Adipose Tissue"

Article Title: Ketogenic Diet Impairs FGF21 Signaling and Promotes Differential Inflammatory Responses in the Liver and White Adipose Tissue

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126364

Ketogenic diet (KD) impairs FGF21 signaling in the liver. A) KD does not modify Klb (β-Klotho) mRNA expression, but downregulates B) Fgfr4 mRNA expression, with a subsequent reduction of C) phospho-ERK1/2 protein levels. Data are presented as means ± SEM. * P
Figure Legend Snippet: Ketogenic diet (KD) impairs FGF21 signaling in the liver. A) KD does not modify Klb (β-Klotho) mRNA expression, but downregulates B) Fgfr4 mRNA expression, with a subsequent reduction of C) phospho-ERK1/2 protein levels. Data are presented as means ± SEM. * P

Techniques Used: Expressing

Ketogenic diet (KD) impairs FGF21 signaling in epididymal white adipose tissue. A) KD does not affect adiponectin plasma levels; KD downregulates B) Adiponectin , C) Klb (β-Klotho), D) Fgfr1 mRNA expression, and E) phosho-ERK1/2 protein levels in white adipose tissue. Data are presented as means ± SEM. * P
Figure Legend Snippet: Ketogenic diet (KD) impairs FGF21 signaling in epididymal white adipose tissue. A) KD does not affect adiponectin plasma levels; KD downregulates B) Adiponectin , C) Klb (β-Klotho), D) Fgfr1 mRNA expression, and E) phosho-ERK1/2 protein levels in white adipose tissue. Data are presented as means ± SEM. * P

Techniques Used: Expressing

52) Product Images from "Insulin Regulates Glucagon-Like Peptide-1 Secretion from the Enteroendocrine L Cell"

Article Title: Insulin Regulates Glucagon-Like Peptide-1 Secretion from the Enteroendocrine L Cell

Journal: Endocrinology

doi: 10.1210/en.2008-0726

Effects of insulin on FRIC cultures. A and B, FRIC cultures were treated with10 −7 m insulin for 5 min, and cell lysates were collected for analysis of Akt (n =4/group) (A) and ERK1/2 (n = 4/group) (B) phosphorylation by Western blot analysis(WB).
Figure Legend Snippet: Effects of insulin on FRIC cultures. A and B, FRIC cultures were treated with10 −7 m insulin for 5 min, and cell lysates were collected for analysis of Akt (n =4/group) (A) and ERK1/2 (n = 4/group) (B) phosphorylation by Western blot analysis(WB).

Techniques Used: Western Blot

Effect of PI3 kinase-Akt and MEK-ERK1/2 inhibition on insulin-mediated GLP-1secretion. GLUTag (A and C) and NCI-H716 (B and D) cells were pretreated with 50μ m LY294002 (A, n = 4–5/group; B, n = 6/group) or 50 μ m PD98059(C, n
Figure Legend Snippet: Effect of PI3 kinase-Akt and MEK-ERK1/2 inhibition on insulin-mediated GLP-1secretion. GLUTag (A and C) and NCI-H716 (B and D) cells were pretreated with 50μ m LY294002 (A, n = 4–5/group; B, n = 6/group) or 50 μ m PD98059(C, n

Techniques Used: Inhibition

53) Product Images from "The ?7nACh-NMDA receptor complex is involved in cue-induced reinstatement of nicotine seeking"

Article Title: The ?7nACh-NMDA receptor complex is involved in cue-induced reinstatement of nicotine seeking

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121270

TAT-α7pep2 treatment blocks cue-induced reinstatement and ERK1/2 phosphorylation. (A and B) Active (A) and inactive (B) lever presses on the last day of extinction and on the test day for nicotine reinstatement. On the reinstatement test day, rats were tested in the presence of the response-contingent light + tone cue after injection with 40 nmol TAT or 12 and 40 nmol TAT-α7pep2[L 336 -M 345 ] 1 h before testing. “#” indicates significant difference from control (TAT) on the test day (P
Figure Legend Snippet: TAT-α7pep2 treatment blocks cue-induced reinstatement and ERK1/2 phosphorylation. (A and B) Active (A) and inactive (B) lever presses on the last day of extinction and on the test day for nicotine reinstatement. On the reinstatement test day, rats were tested in the presence of the response-contingent light + tone cue after injection with 40 nmol TAT or 12 and 40 nmol TAT-α7pep2[L 336 -M 345 ] 1 h before testing. “#” indicates significant difference from control (TAT) on the test day (P

Techniques Used: Injection

54) Product Images from "Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status"

Article Title: Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-75

Downstream PI3K and MAPK pathway activation. Melanoma cells were starved in 0.2% FBS overnight and then protein lysates were collected and evaluated by Western blot analysis for activation of ERK1/2 and Akt. Numerical values for the IC50 of E6201 for each cell line, as well as phosphorylation status scoring for both ERK1/2 and Akt is provided in tabular form.
Figure Legend Snippet: Downstream PI3K and MAPK pathway activation. Melanoma cells were starved in 0.2% FBS overnight and then protein lysates were collected and evaluated by Western blot analysis for activation of ERK1/2 and Akt. Numerical values for the IC50 of E6201 for each cell line, as well as phosphorylation status scoring for both ERK1/2 and Akt is provided in tabular form.

Techniques Used: Activation Assay, Western Blot

55) Product Images from "Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro"

Article Title: Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro

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

doi: 10.12659/MSM.919089

The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P
Figure Legend Snippet: The ERK inhibitor, PD98059, blocked the oncogenic effects of silencing dual-specificity phosphatase 2 (DUSP2) in SKOV3 and OVCAR3 cells in vitro . SKOV3 and OVCAR3 cells transfected with DUSP2 siRNA were further treated with either 25 μM of the ERK1/2 inhibitor (PD98059) or with dimethyl sulfoxide (DMSO) (control). The cell proliferation and migration capacities were evaluated by the cell counting kit-8 (CCK-8) assay ( A, B ) and wound-healing assay ( C, D ), respectively. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

Techniques Used: In Vitro, Transfection, Migration, Cell Counting, CCK-8 Assay, Wound Healing Assay, Standard Deviation

Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P
Figure Legend Snippet: Dual-specificity phosphatase 2 (DUSP2) inhibited ERK1/2 activation and cell proliferation of SKOV3 and OVCAR3 cells in vitro. ( A ) The protein expression level of DUSP2 in OSE, SKOV3, and OVCAR3 cells were compared by Western blot. DUSP2 was down-regulated in SKOV3 and OVCAR3 cell lines. ( B ) Both SKOV3 and OVCAR3 cells were transfected with either siRNA targeting DUSP2 or plasmid overexpressing DUSP2. The transfection efficiency was evaluated by Western blot and compared with the control cells treated with transfection reagents. ( C, D ) The proliferation capacity of SKOV3 and OVCAR3 cells was estimated by the cell counting kit-8 (CCK-8) assay. ( E, F ) The wound-healing assay was conducted to evaluate the effects of silencing or overexpression of DUSP2 on cell migration. Data are shown as the mean±standard deviation (SD) from three independent experiments (* P

Techniques Used: Activation Assay, In Vitro, Expressing, Western Blot, Transfection, Plasmid Preparation, Cell Counting, CCK-8 Assay, Wound Healing Assay, Over Expression, Migration, Standard Deviation

56) Product Images from "Autophagy Induced FHL2 Upregulation Promotes IL-6 Production by Activating the NF-κB Pathway in Mouse Aortic Endothelial Cells after Exposure to PM2.5"

Article Title: Autophagy Induced FHL2 Upregulation Promotes IL-6 Production by Activating the NF-κB Pathway in Mouse Aortic Endothelial Cells after Exposure to PM2.5

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18071484

Activation of NF-κB and FHL2 upregulation by PM2.5. ( A ) Cells were incubated with 0, 12.5, 25, 50, and 100 µg/mL PM2.5 for 24 h, and phosphorylated p65 (p-p65), p65, phosphorylated IκBα (p-IκBα), IκBα, phosphorylated p38(p-p38), p38, phosphorylated Akt (p-Akt), Akt, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, FHL2 , and β-actin were detected in PM2.5-treated mouse aortic endothelial cells (MAECs). ( n = 3); ( B – E ) Band quantifications for western blot analysis. * p
Figure Legend Snippet: Activation of NF-κB and FHL2 upregulation by PM2.5. ( A ) Cells were incubated with 0, 12.5, 25, 50, and 100 µg/mL PM2.5 for 24 h, and phosphorylated p65 (p-p65), p65, phosphorylated IκBα (p-IκBα), IκBα, phosphorylated p38(p-p38), p38, phosphorylated Akt (p-Akt), Akt, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, FHL2 , and β-actin were detected in PM2.5-treated mouse aortic endothelial cells (MAECs). ( n = 3); ( B – E ) Band quantifications for western blot analysis. * p

Techniques Used: Activation Assay, Incubation, Western Blot

57) Product Images from "miR-429 suppresses tumor migration and invasion by targeting CRKL in hepatocellular carcinoma via inhibiting Raf/MEK/ERK pathway and epithelial-mesenchymal transition"

Article Title: miR-429 suppresses tumor migration and invasion by targeting CRKL in hepatocellular carcinoma via inhibiting Raf/MEK/ERK pathway and epithelial-mesenchymal transition

Journal: Scientific Reports

doi: 10.1038/s41598-018-20258-8

The influences of miR-429 and CRKL on Raf/MEK/ERK pathway and EMT in HepG2 cells. ( A ) Comparative analysis of Ras, Raf, p-Raf, p-MEK, ERK1/2 and p-ERK2 protein levels in HepG2 cells transfected with miR-429 mimic, miR-429 inhibitor, shCRKL or PCDH-CRKL. ( B – E ) Comparative analysis of mRNA levels of E-cadherin, N-cadherin, Vimentin and c-Jun in above group HepG2 cells. * , ** and *** refer to the level changes were in statistical significances with P
Figure Legend Snippet: The influences of miR-429 and CRKL on Raf/MEK/ERK pathway and EMT in HepG2 cells. ( A ) Comparative analysis of Ras, Raf, p-Raf, p-MEK, ERK1/2 and p-ERK2 protein levels in HepG2 cells transfected with miR-429 mimic, miR-429 inhibitor, shCRKL or PCDH-CRKL. ( B – E ) Comparative analysis of mRNA levels of E-cadherin, N-cadherin, Vimentin and c-Jun in above group HepG2 cells. * , ** and *** refer to the level changes were in statistical significances with P

Techniques Used: Transfection

58) Product Images from "Tubeimoside I Protects Dopaminergic Neurons Against Inflammation-Mediated Damage in Lipopolysaccharide (LPS)-Evoked Model of Parkinson’s Disease in Rats"

Article Title: Tubeimoside I Protects Dopaminergic Neurons Against Inflammation-Mediated Damage in Lipopolysaccharide (LPS)-Evoked Model of Parkinson’s Disease in Rats

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082242

TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p
Figure Legend Snippet: TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p

Techniques Used: Incubation, Western Blot

59) Product Images from "Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *"

Article Title: Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.188854

NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific
Figure Legend Snippet: NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific

Techniques Used:

NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,

Techniques Used: Activation Assay, Injection

NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free
Figure Legend Snippet: NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free

Techniques Used: Activation Assay, Lactate Dehydrogenase Assay

NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (
Figure Legend Snippet: NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (

Techniques Used: Activation Assay

NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations

Techniques Used: Activation Assay, In Vitro

60) Product Images from "Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *"

Article Title: Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.188854

NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific
Figure Legend Snippet: NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific

Techniques Used:

NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,

Techniques Used: Activation Assay, Injection

NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free
Figure Legend Snippet: NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free

Techniques Used: Activation Assay, Lactate Dehydrogenase Assay

NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (
Figure Legend Snippet: NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (

Techniques Used: Activation Assay

NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations

Techniques Used: Activation Assay, In Vitro

61) Product Images from "Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *"

Article Title: Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.188854

NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific
Figure Legend Snippet: NMDA-induced neurotoxicity in mature neuron is p38 MAPK pathway-dependent, whereas neuroprotection in immature neuron is ERK1/2-dependent. At 12 or 3DIV, neurons were pretreated with p38 MAPK-specific inhibitor SB203580 (10 μ m ), ERK1/2-specific

Techniques Used:

NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in adult rat hippocampus, although it favors ERK1/2 activation in P0 rat hippocampus. A and B , NMDA (60 m m , 1 μl) or PBS (control) was administered to adult or P0 rat ( n = 6) by i.c.v. injection. After 30 min,

Techniques Used: Activation Assay, Injection

NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free
Figure Legend Snippet: NMDA neurotoxicity but not NMDA induced ERK1/2 and p38 MAPK activation changes according to extracellular calcium level. For LDH release assay, 3- and 12DIV neurons were treated with or without NMDA (100 μ m ) for 15 min in normal or calcium-free

Techniques Used: Activation Assay, Lactate Dehydrogenase Assay

NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (
Figure Legend Snippet: NR2B-containing NMDARs mediate both p38 MAPK activation in mature neuron and ERK1/2 activation in immature neuron. At 3 or 12DIV, neurons were starved for 6 h in DMEM without any supplement and were pretreated with MK-801 (0.3 μ m ), Ro-256981 (

Techniques Used: Activation Assay

NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations
Figure Legend Snippet: NMDA prefers to activate p38 MAPK in mature neurons, whereas it favors ERK1/2 activation in immature neurons. On different days in vitro , neurons were starved for 6 h in DMEM without any supplement and then stimulated with the indicated concentrations

Techniques Used: Activation Assay, In Vitro

62) Product Images from "Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C) *"

Article Title: Lymphatic Endothelial Heparan Sulfate Deficiency Results in Altered Growth Responses to Vascular Endothelial Growth Factor-C (VEGF-C) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.206664

Silencing of lymphatic endothelial heparan sulfate chain biosynthesis inhibits VEGF-C-dependent Erk1/2 activation and reduces receptor-dependent binding of VEGF-C. A , cultured primary human lymphatic endothelia were transfected with siRNA targeting XylT2
Figure Legend Snippet: Silencing of lymphatic endothelial heparan sulfate chain biosynthesis inhibits VEGF-C-dependent Erk1/2 activation and reduces receptor-dependent binding of VEGF-C. A , cultured primary human lymphatic endothelia were transfected with siRNA targeting XylT2

Techniques Used: Activation Assay, Binding Assay, Cell Culture, Transfection

VEGF-C-mediated Erk1/2 activation in lymphatic endothelium is inhibited by the presence of heparin, with unique responses in the presence of sulfate-modified heparinoids. A , phosphorylation of Erk1/2 (p44/42) in serum-starved human primary lung lymphatic
Figure Legend Snippet: VEGF-C-mediated Erk1/2 activation in lymphatic endothelium is inhibited by the presence of heparin, with unique responses in the presence of sulfate-modified heparinoids. A , phosphorylation of Erk1/2 (p44/42) in serum-starved human primary lung lymphatic

Techniques Used: Activation Assay, Modification

Heparinase-mediated ablation of lymphatic endothelial cell-surface heparan sulfate inhibits growth signaling and receptor activation in response to VEGF-C. A , phosphorylation of Erk1/2 (p44/42) in serum-starved primary human lung lymphatic endothelial
Figure Legend Snippet: Heparinase-mediated ablation of lymphatic endothelial cell-surface heparan sulfate inhibits growth signaling and receptor activation in response to VEGF-C. A , phosphorylation of Erk1/2 (p44/42) in serum-starved primary human lung lymphatic endothelial

Techniques Used: Activation Assay

63) Product Images from "DiGeorge Syndrome Critical Region 8 (DGCR8) Protein-mediated microRNA Biogenesis Is Essential for Vascular Smooth Muscle Cell Development in Mice *"

Article Title: DiGeorge Syndrome Critical Region 8 (DGCR8) Protein-mediated microRNA Biogenesis Is Essential for Vascular Smooth Muscle Cell Development in Mice *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.351791

Loss of DGCR8 in VSMCs leads to attenuation of ERK1/2 and pAKT in umbilical arteries and VSMC KO cells. A , phospho and total ERK1/2 and AKT were detected by Western blot analysis in umbilical arteries pooled from embryos of DGCR8 cKOs and WT controls
Figure Legend Snippet: Loss of DGCR8 in VSMCs leads to attenuation of ERK1/2 and pAKT in umbilical arteries and VSMC KO cells. A , phospho and total ERK1/2 and AKT were detected by Western blot analysis in umbilical arteries pooled from embryos of DGCR8 cKOs and WT controls

Techniques Used: Western Blot

64) Product Images from "Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas"

Article Title: Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0502070102

Differential activation of p -ERK1/2-ir in hippocampal neurons after vehicle or i.c.v. CRH injections. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained as described in Methods . Pyramidal
Figure Legend Snippet: Differential activation of p -ERK1/2-ir in hippocampal neurons after vehicle or i.c.v. CRH injections. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained as described in Methods . Pyramidal

Techniques Used: Activation Assay, Mouse Assay, Immunohistochemistry, Staining

Quantitative analysis of p -ERK1/2-ir. Levels of p -ERK1/2 in CA3, CA1, and the BLC (BLC = LA and BLA) 10 min ( a ) and 30 min ( b ) after i.c.v. vehicle or CRH administration. Optical density data processed with the fluoview acquisition/analyzer program are
Figure Legend Snippet: Quantitative analysis of p -ERK1/2-ir. Levels of p -ERK1/2 in CA3, CA1, and the BLC (BLC = LA and BLA) 10 min ( a ) and 30 min ( b ) after i.c.v. vehicle or CRH administration. Optical density data processed with the fluoview acquisition/analyzer program are

Techniques Used:

CRH induces p -ERK1/2 levels in the amygdala but not in the cortex or hypothalamus. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained 30 min after i.c.v. CRH injections as described
Figure Legend Snippet: CRH induces p -ERK1/2 levels in the amygdala but not in the cortex or hypothalamus. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained 30 min after i.c.v. CRH injections as described

Techniques Used: Mouse Assay, Immunohistochemistry, Staining

Quantitative analysis of p -ERK1/2-ir in Crhr1 loxP/loxP control and Crhr1 loxP/loxP Camk2a-cre mice after central CRH administration. Levels of p -ERK1/2 in CA3, CA1, and the BLC (BLC = LA and BLA) of the amygdala 10 min ( a ) and 30 min ( b ) after i.c.v. CRH
Figure Legend Snippet: Quantitative analysis of p -ERK1/2-ir in Crhr1 loxP/loxP control and Crhr1 loxP/loxP Camk2a-cre mice after central CRH administration. Levels of p -ERK1/2 in CA3, CA1, and the BLC (BLC = LA and BLA) of the amygdala 10 min ( a ) and 30 min ( b ) after i.c.v. CRH

Techniques Used: Mouse Assay

Crhr1 loxP/loxP Camk2a-cre mice show a decreased ERK1/2 activation response in CA3, CA1, and the BLC after central CRH administration. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained
Figure Legend Snippet: Crhr1 loxP/loxP Camk2a-cre mice show a decreased ERK1/2 activation response in CA3, CA1, and the BLC after central CRH administration. Representative images (from five mice) of p -ERK1/2 immunohistochemistry ( Upper ) and control PI staining ( Lower ) obtained

Techniques Used: Mouse Assay, Activation Assay, Immunohistochemistry, Staining

65) Product Images from "Cholangiocyte N-Ras Protein Mediates Lipopolysaccharide-induced Interleukin 6 Secretion and Proliferation *"

Article Title: Cholangiocyte N-Ras Protein Mediates Lipopolysaccharide-induced Interleukin 6 Secretion and Proliferation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.269464

LPS induces IL6 expression in an N-Ras-dependent manner. A , H69 cells transfected with either the scrambled ( Scr ) or N-Ras siRNA were cultured in the presence or absence of LPS (200 ng/ml) for 6 h, and IL6 immunoblotting was performed. IL6 expression was induced in the cells transfected with the scrambled siRNA; however, cells depleted of N-Ras exhibited no increase in IL6 expression at this time point. Actin was blotted as a loading control. B , cholangiocytes were treated with LPS (200 ng/ml) for 15 min, washed, and cultured over the course of 6 h. An IL6 ELISA was performed on tissue culture supernatant, and an N-Ras activation assay was performed on the cellular lysate ( n = 3). N-Ras activation exhibited a biphasic response to LPS-treatment. Activation of N-Ras occurred immediately following LPS exposure (15 min), decreased at 1 h, and increased again after 3 h. In contrast, an increase in IL6 secretion was first detected 1 h after LPS treatment and was consistently elevated from 3 h after LPS treatment. C , the cellular lysate from each time point was utilized for an N-Ras activation assay and immunoblotting for phosphorylated ERK1/2, total ERK1/2 (loading control), and phosphorylated STAT3. N-Ras activation was observed at the 15-min time point and correlated with the phosphorylation of ERK1/2. In contrast, STAT3 phosphorylation was delayed (1 h) and remained elevated through 6-h after LPS treatment. D , an IL6 receptor inhibitory antibody ( IL6R Ab ) diminishes N-Ras activation at the 6-h time point. Cells were cultured in the presence or absence of an IL6 inhibitory antibody and in the presence or absence of LPS. The IL6-inhibiting antibody did not inhibit N-Ras activation 15 min after LPS treatment, but diminished N-Ras activation at the 6-h time point.
Figure Legend Snippet: LPS induces IL6 expression in an N-Ras-dependent manner. A , H69 cells transfected with either the scrambled ( Scr ) or N-Ras siRNA were cultured in the presence or absence of LPS (200 ng/ml) for 6 h, and IL6 immunoblotting was performed. IL6 expression was induced in the cells transfected with the scrambled siRNA; however, cells depleted of N-Ras exhibited no increase in IL6 expression at this time point. Actin was blotted as a loading control. B , cholangiocytes were treated with LPS (200 ng/ml) for 15 min, washed, and cultured over the course of 6 h. An IL6 ELISA was performed on tissue culture supernatant, and an N-Ras activation assay was performed on the cellular lysate ( n = 3). N-Ras activation exhibited a biphasic response to LPS-treatment. Activation of N-Ras occurred immediately following LPS exposure (15 min), decreased at 1 h, and increased again after 3 h. In contrast, an increase in IL6 secretion was first detected 1 h after LPS treatment and was consistently elevated from 3 h after LPS treatment. C , the cellular lysate from each time point was utilized for an N-Ras activation assay and immunoblotting for phosphorylated ERK1/2, total ERK1/2 (loading control), and phosphorylated STAT3. N-Ras activation was observed at the 15-min time point and correlated with the phosphorylation of ERK1/2. In contrast, STAT3 phosphorylation was delayed (1 h) and remained elevated through 6-h after LPS treatment. D , an IL6 receptor inhibitory antibody ( IL6R Ab ) diminishes N-Ras activation at the 6-h time point. Cells were cultured in the presence or absence of an IL6 inhibitory antibody and in the presence or absence of LPS. The IL6-inhibiting antibody did not inhibit N-Ras activation 15 min after LPS treatment, but diminished N-Ras activation at the 6-h time point.

Techniques Used: Expressing, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay

66) Product Images from "CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING"

Article Title: CAVEOLIN-1 REGULATES HIV-1 TAT-INDUCED ALTERATIONS OF TIGHT JUNCTION PROTEIN EXPRESSION VIA MODULATION OF THE RAS SIGNALING

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.0061-08.2008

Tat-mediated activation of ERK1/2 in HBMEC is downstream from Ras and MEK1/2
Figure Legend Snippet: Tat-mediated activation of ERK1/2 in HBMEC is downstream from Ras and MEK1/2

Techniques Used: Activation Assay

67) Product Images from "The dual-inhibitory effect of miR-338-5p on the multidrug resistance and cell growth of hepatocellular carcinoma"

Article Title: The dual-inhibitory effect of miR-338-5p on the multidrug resistance and cell growth of hepatocellular carcinoma

Journal: Signal Transduction and Targeted Therapy

doi: 10.1038/s41392-017-0003-4

miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway. a The expression levels of P-gp were determined by western blotting after transfection with EGFR siRNA. b P-gp was increased in cells treated with the EGFR vector. c – d The P-gp levels in cells after co-transfection with miR-338-5p and EGFR vector. Western blot showing expression levels of p-EGFR, ERK1/2, and p-ERK1/2 in cells transfected with miR-338-5p mimics e – f or inhibitors g – h . i – j HCC cells were treated with 15 μM U0126 (an inhibitor of ERK1/2 signaling) for 24 h, and then, the protein levels of genes were detected by western blot analysis. Student’s t -test (two-tailed) or one-way analysis of variance was employed to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. ** P
Figure Legend Snippet: miR-338-5p inhibits ABCB1 expression by targeting the EGFR/ERK1/2 signaling pathway. a The expression levels of P-gp were determined by western blotting after transfection with EGFR siRNA. b P-gp was increased in cells treated with the EGFR vector. c – d The P-gp levels in cells after co-transfection with miR-338-5p and EGFR vector. Western blot showing expression levels of p-EGFR, ERK1/2, and p-ERK1/2 in cells transfected with miR-338-5p mimics e – f or inhibitors g – h . i – j HCC cells were treated with 15 μM U0126 (an inhibitor of ERK1/2 signaling) for 24 h, and then, the protein levels of genes were detected by western blot analysis. Student’s t -test (two-tailed) or one-way analysis of variance was employed to analyze the data. Data in all experiments are presented as the means ± SD of three independent experiments. ** P

Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation, Cotransfection, Two Tailed Test

68) Product Images from "Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition"

Article Title: Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

Journal: Cancer Cell International

doi: 10.1186/1475-2867-11-44

The multi-kinase inhibitor sorafenib induces alterations in cellular signaling and apoptosis regulators in AT/RT cells in response to conditioned medium . Addition of autologous conditioned medium to briefly serum starved cells provides an avenue to evaluate the potential responses that may occur during autocrine/paracrine growth stimulation. In the presence of sorafenib (S), the conditioned medium induced reduced phosphorylation of Erk1/2 (BT12, KCCF1), Akt1/2 (BT12), c-Raf (BT12), Stat3 (BT12, KCCF1) compared to DMSO (D) control. Under these conditions, an increase in phosphorylation of c-Raf was noted in KCCF1 cells. However, the pro-survival protein Mcl-1 was suppressed by sorafenib in all three cell lines (A). Compared to basal medium (BM), conditioned medium (CM) induced phosphorylation of signaling regulators, such as Erk (B). The data shown are typical of three separate experiments.
Figure Legend Snippet: The multi-kinase inhibitor sorafenib induces alterations in cellular signaling and apoptosis regulators in AT/RT cells in response to conditioned medium . Addition of autologous conditioned medium to briefly serum starved cells provides an avenue to evaluate the potential responses that may occur during autocrine/paracrine growth stimulation. In the presence of sorafenib (S), the conditioned medium induced reduced phosphorylation of Erk1/2 (BT12, KCCF1), Akt1/2 (BT12), c-Raf (BT12), Stat3 (BT12, KCCF1) compared to DMSO (D) control. Under these conditions, an increase in phosphorylation of c-Raf was noted in KCCF1 cells. However, the pro-survival protein Mcl-1 was suppressed by sorafenib in all three cell lines (A). Compared to basal medium (BM), conditioned medium (CM) induced phosphorylation of signaling regulators, such as Erk (B). The data shown are typical of three separate experiments.

Techniques Used:

69) Product Images from "Inhibition of (pro)renin Receptor Contributes to Renoprotective Effects of Angiotensin II Type 1 Receptor Blockade in Diabetic Nephropathy"

Article Title: Inhibition of (pro)renin Receptor Contributes to Renoprotective Effects of Angiotensin II Type 1 Receptor Blockade in Diabetic Nephropathy

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2017.00758

Schematic diagram of a proposed mechanism for blockade of (pro)renin/PRR activation as a therapeutic strategy. Binding of (pro)renin and PRR activates ERK1/2 signaling, resulting in the production of profibrotic factors in diabetic nephropathy, such as TGF-β1. During diabetes development, high glucose and Ang II, as well as high level (pro)renin, lead to a downregulation of PRR expression, through the mediation of AT 2 R (He et al., 2010 ) or PLZF (Schefe et al., 2006 ), indicating a negative feedback mechanism. But, the activation of the downregulated PRR still involes in the diabetic nephropathy development. Besides blocking AT 1 R, losartan plays renoprotective roles through further suppressing PRR via upregulation of AT 2 R. Moreover, HRP may enhance losartan's anti-fibrotic effects through further or fully inhibiting PRR. The present findings highlight blockade of PRR as a possible new addition therapy to AT 1 R blockade for DN. Abbreviations: Ang II, angiotensin II; AT 1 R, angiotensin II type 1 receptor; AT 2 R, angiotensin II type 2 receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; HRP, handle region peptide (HRP) of prorenin; PLZF, promyelocytic zinc finger; PRR, (pro)renin receptor; TGF-β1, transforming growth factor-β1.
Figure Legend Snippet: Schematic diagram of a proposed mechanism for blockade of (pro)renin/PRR activation as a therapeutic strategy. Binding of (pro)renin and PRR activates ERK1/2 signaling, resulting in the production of profibrotic factors in diabetic nephropathy, such as TGF-β1. During diabetes development, high glucose and Ang II, as well as high level (pro)renin, lead to a downregulation of PRR expression, through the mediation of AT 2 R (He et al., 2010 ) or PLZF (Schefe et al., 2006 ), indicating a negative feedback mechanism. But, the activation of the downregulated PRR still involes in the diabetic nephropathy development. Besides blocking AT 1 R, losartan plays renoprotective roles through further suppressing PRR via upregulation of AT 2 R. Moreover, HRP may enhance losartan's anti-fibrotic effects through further or fully inhibiting PRR. The present findings highlight blockade of PRR as a possible new addition therapy to AT 1 R blockade for DN. Abbreviations: Ang II, angiotensin II; AT 1 R, angiotensin II type 1 receptor; AT 2 R, angiotensin II type 2 receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; HRP, handle region peptide (HRP) of prorenin; PLZF, promyelocytic zinc finger; PRR, (pro)renin receptor; TGF-β1, transforming growth factor-β1.

Techniques Used: Activation Assay, Binding Assay, Expressing, Blocking Assay

HRP suppressed high glucose and Ang II-induced proliferative and fibrotic effects in MCs. Antagonistic action of HRP (10 −7 M and 10 −6 M) on high glucose-induced increase in phosphorylation of ERK1/2 (A) and TGF-β1 mRNA expression (B) by real-time PCR. (C) Abolition of the high glucose-induced decrease in MMP-2 activity by HRP by gelatin zymography. * P
Figure Legend Snippet: HRP suppressed high glucose and Ang II-induced proliferative and fibrotic effects in MCs. Antagonistic action of HRP (10 −7 M and 10 −6 M) on high glucose-induced increase in phosphorylation of ERK1/2 (A) and TGF-β1 mRNA expression (B) by real-time PCR. (C) Abolition of the high glucose-induced decrease in MMP-2 activity by HRP by gelatin zymography. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Zymography

70) Product Images from "Angiotensin-converting enzyme 2 activator diminazene aceturate prevents lipopolysaccharide-induced inflammation by inhibiting MAPK and NF-κB pathways in human retinal pigment epithelium"

Article Title: Angiotensin-converting enzyme 2 activator diminazene aceturate prevents lipopolysaccharide-induced inflammation by inhibiting MAPK and NF-κB pathways in human retinal pigment epithelium

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-016-0489-7

Effect of DIZE on the phosphorylation levels of MAPKs and the activation of NF-κB in ARPE-19 cells stimulated with LPS. a Western blotting was used to determine the protein levels of p38 MAPK, JNK, ERK1/2 and their phosphorylated forms (p-p38, p-JNK, and p-ERK1/2). b Activation of NF-κB pathway was indicated by p-IκB-α, an indicator for the activation of NF-κB. The expressions of p-p38, p-JNK, and p-ERK1/2 were normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE treatment resulted in remarkably reduction of phosphorylation of p38, JNK, ERK1/2, and IκB-α compared with the LPS-stimulated group. All data are expressed as mean ± SEM (*** p
Figure Legend Snippet: Effect of DIZE on the phosphorylation levels of MAPKs and the activation of NF-κB in ARPE-19 cells stimulated with LPS. a Western blotting was used to determine the protein levels of p38 MAPK, JNK, ERK1/2 and their phosphorylated forms (p-p38, p-JNK, and p-ERK1/2). b Activation of NF-κB pathway was indicated by p-IκB-α, an indicator for the activation of NF-κB. The expressions of p-p38, p-JNK, and p-ERK1/2 were normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE treatment resulted in remarkably reduction of phosphorylation of p38, JNK, ERK1/2, and IκB-α compared with the LPS-stimulated group. All data are expressed as mean ± SEM (*** p

Techniques Used: Activation Assay, Western Blot

Effect of the MAPK and NF-κB inhibitors on the mRNA and protein expression of inflammatory cytokines in LPS-stimulated ARPE-19 cells. The expression of IL-6, IL-8, and MCP-1 was detected by real-time PCR and ELISA assay in the MAPK pathway inhibitor-treated cells ( a ) and NF-κB pathway inhibitor-treated cells ( b ). The p38 inhibitor (SB203580, SB), JNK inhibitor (SP600125, SP), and ERK1/2 inhibitor (PD98059, PD) significantly decreased the production of IL-6, IL-8, and MCP-1 in the ARPE-19 cells stimulated with LPS at both mRNA and protein levels ( a ). The NF-κB inhibitor (BAY 11-7082, BAY) remarkably reduced the production of IL-6, IL-8, and MCP-1 in LPS-treated group at both mRNA and protein levels ( b ). All data are expressed as mean ± SEM (*** p
Figure Legend Snippet: Effect of the MAPK and NF-κB inhibitors on the mRNA and protein expression of inflammatory cytokines in LPS-stimulated ARPE-19 cells. The expression of IL-6, IL-8, and MCP-1 was detected by real-time PCR and ELISA assay in the MAPK pathway inhibitor-treated cells ( a ) and NF-κB pathway inhibitor-treated cells ( b ). The p38 inhibitor (SB203580, SB), JNK inhibitor (SP600125, SP), and ERK1/2 inhibitor (PD98059, PD) significantly decreased the production of IL-6, IL-8, and MCP-1 in the ARPE-19 cells stimulated with LPS at both mRNA and protein levels ( a ). The NF-κB inhibitor (BAY 11-7082, BAY) remarkably reduced the production of IL-6, IL-8, and MCP-1 in LPS-treated group at both mRNA and protein levels ( b ). All data are expressed as mean ± SEM (*** p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Effect of A779 on the phosphorylation of MAPKs and the activation of NF-κB in ARPE-19. Western blotting was applied to determine the protein levels of p-p38, p-JNK, p-ERK1/2, p38, JNK, ERK1/2 ( a ) and p-IκB-α, an indicator for the activation of NF-κB ( b ). The band intensity of p-p38, p-JNK, and p-ERK1/2 was normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE reduced the phosphorylation of p38, JNK, and ERK1/2 and IκB-α induced by LPS, while A779 abrogated DIZE’s effect. All data are expressed as mean ± SEM (*** p
Figure Legend Snippet: Effect of A779 on the phosphorylation of MAPKs and the activation of NF-κB in ARPE-19. Western blotting was applied to determine the protein levels of p-p38, p-JNK, p-ERK1/2, p38, JNK, ERK1/2 ( a ) and p-IκB-α, an indicator for the activation of NF-κB ( b ). The band intensity of p-p38, p-JNK, and p-ERK1/2 was normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE reduced the phosphorylation of p38, JNK, and ERK1/2 and IκB-α induced by LPS, while A779 abrogated DIZE’s effect. All data are expressed as mean ± SEM (*** p

Techniques Used: Activation Assay, Western Blot

Effect of ACE2-siRNA on the phosphorylation of MAPKs and the activation of NF-κB in ARPE-19. The protein levels of p-p38, p-JNK, p-ERK1/2, p38, JNK, ERK1/2 ( a ) and p-IκB-α ( b ) were determined by Western blotting. The band intensity of p-p38, p-JNK, and p-ERK1/2 was normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE reduced the phosphorylation of p38, JNK, and ERK1/2 and IκB-α induced by LPS, while ACE2-siRNA abolished DIZE’s effect. All data are expressed as mean ± SEM (*** p
Figure Legend Snippet: Effect of ACE2-siRNA on the phosphorylation of MAPKs and the activation of NF-κB in ARPE-19. The protein levels of p-p38, p-JNK, p-ERK1/2, p38, JNK, ERK1/2 ( a ) and p-IκB-α ( b ) were determined by Western blotting. The band intensity of p-p38, p-JNK, and p-ERK1/2 was normalized to p38, JNK, and ERK1/2, respectively. The band intensity of p-IκB-α was normalized to β-actin. DIZE reduced the phosphorylation of p38, JNK, and ERK1/2 and IκB-α induced by LPS, while ACE2-siRNA abolished DIZE’s effect. All data are expressed as mean ± SEM (*** p

Techniques Used: Activation Assay, Western Blot

71) Product Images from "Ischemic Preconditioning VIA Epsilon PKC Activation Requires Cyclooxygenase-2 Activation in Vitro"

Article Title: Ischemic Preconditioning VIA Epsilon PKC Activation Requires Cyclooxygenase-2 Activation in Vitro

Journal:

doi: 10.1016/j.neuroscience.2006.12.063

εPKC-mediated neuroprotection prevented by inhibition of ERK1/2 and COX-2 activation. 10 μM PD98059 was treated during 1 h of εPKC-activating peptide treatment (ψεRACK at 100 nM) and 48 h of reperfusion, or NS-398
Figure Legend Snippet: εPKC-mediated neuroprotection prevented by inhibition of ERK1/2 and COX-2 activation. 10 μM PD98059 was treated during 1 h of εPKC-activating peptide treatment (ψεRACK at 100 nM) and 48 h of reperfusion, or NS-398

Techniques Used: Inhibition, Activation Assay

εPKC activation induced ERK1/2 phosphorylation and Inhibition of ERK1/2 phosphorylation reduced IPC-mediated neuroprotection and COX-2 expression in mixed cortical neuron/astrocyte cell cultures. (A) Representative western blots of ERK1/2 protein
Figure Legend Snippet: εPKC activation induced ERK1/2 phosphorylation and Inhibition of ERK1/2 phosphorylation reduced IPC-mediated neuroprotection and COX-2 expression in mixed cortical neuron/astrocyte cell cultures. (A) Representative western blots of ERK1/2 protein

Techniques Used: Activation Assay, Inhibition, Expressing, Western Blot

72) Product Images from "Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞"

Article Title: Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M709914200

Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.
Figure Legend Snippet: Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.

Techniques Used: Migration

73) Product Images from "Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞"

Article Title: Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? *Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1? * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M709914200

Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.
Figure Legend Snippet: Proposed mechanisms by which NO mediates epithelial cell migration through regulation of ERK1/2 and HIF-1α pathways. VEGF , vascular endothelial growth factor.

Techniques Used: Migration

74) Product Images from "MEK inhibition suppresses cell invasion and migration in ovarian cancers with activation of ERK1/2"

Article Title: MEK inhibition suppresses cell invasion and migration in ovarian cancers with activation of ERK1/2

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm_00000093

Twist immunoreactivity in ovarian cancer tissues. (A) Intense immunoreactivity is present in the nuclei of carcinoma cells. (B) In this sample negative staining for Twist is noted. (C) Correlation between the p-ERK1/2 immunohistochemical score and the Twist immunohistochemical score in ovarian cancer tissue. The HSCORE for p-ERK1/2 significantly correlates with the Twist immunohistochemical HSCORE (r=0.370, P=0.003).
Figure Legend Snippet: Twist immunoreactivity in ovarian cancer tissues. (A) Intense immunoreactivity is present in the nuclei of carcinoma cells. (B) In this sample negative staining for Twist is noted. (C) Correlation between the p-ERK1/2 immunohistochemical score and the Twist immunohistochemical score in ovarian cancer tissue. The HSCORE for p-ERK1/2 significantly correlates with the Twist immunohistochemical HSCORE (r=0.370, P=0.003).

Techniques Used: Negative Staining, Immunohistochemistry

Western blot analysis. (A) Expression of p-ERK1/2 is undetectable in all CI-1040-treated samples. A similar amount of protein was loaded in CI-1040- and DMSO-treated (control) samples as evidenced by a similar intensity of total ERK1/2. (B) Expression of Twist is undetectable or reduced in all CI-1040-treated samples.
Figure Legend Snippet: Western blot analysis. (A) Expression of p-ERK1/2 is undetectable in all CI-1040-treated samples. A similar amount of protein was loaded in CI-1040- and DMSO-treated (control) samples as evidenced by a similar intensity of total ERK1/2. (B) Expression of Twist is undetectable or reduced in all CI-1040-treated samples.

Techniques Used: Western Blot, Expressing

Immunohistochemical staining of phosphorylated extracellular-regulated kinase (p-ERK)1/2. (A) Intense immunoreactivity is present in both the nucleus and cytoplasm in this ovarian carcinoma. (B) A case showing negative staining of p-ERK1/2. (C) Kaplan-Meier survival curve in 88 patients with ovarian carcinoma according to p-ERK1/2 expression. p-ERK1/2 expression does not correlate with shorter overall survival in patients with ovarian carcinomas.
Figure Legend Snippet: Immunohistochemical staining of phosphorylated extracellular-regulated kinase (p-ERK)1/2. (A) Intense immunoreactivity is present in both the nucleus and cytoplasm in this ovarian carcinoma. (B) A case showing negative staining of p-ERK1/2. (C) Kaplan-Meier survival curve in 88 patients with ovarian carcinoma according to p-ERK1/2 expression. p-ERK1/2 expression does not correlate with shorter overall survival in patients with ovarian carcinomas.

Techniques Used: Immunohistochemistry, Staining, Negative Staining, Expressing

The effects of a MEK inhibitor on cell migration. (A) A simulated wound was created by scraping a confluent monolayer of ES2 cells. Compared to the ES2 cells treated with DMSO, there was a significant reduction in the number of CI-1040-treated ES2 cells that migrated into the wound. (B) Inactivation of p-ERK1/2 affects cell migration in ES2 and MPSC1 (p-ERK1/2-positive) but not in KF28 (p-ERK1/2-negative) cells.
Figure Legend Snippet: The effects of a MEK inhibitor on cell migration. (A) A simulated wound was created by scraping a confluent monolayer of ES2 cells. Compared to the ES2 cells treated with DMSO, there was a significant reduction in the number of CI-1040-treated ES2 cells that migrated into the wound. (B) Inactivation of p-ERK1/2 affects cell migration in ES2 and MPSC1 (p-ERK1/2-positive) but not in KF28 (p-ERK1/2-negative) cells.

Techniques Used: Migration

75) Product Images from "NAD(P)H Oxidase Mediates TGF-?1-Induced Activation of Kidney Myofibroblasts"

Article Title: NAD(P)H Oxidase Mediates TGF-?1-Induced Activation of Kidney Myofibroblasts

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2009020146

Proposed signaling cascade involving Smad3, ROS, and ERK1/2 in TGF-β1–induced kidney myofibroblast activation and matrix synthesis is shown.
Figure Legend Snippet: Proposed signaling cascade involving Smad3, ROS, and ERK1/2 in TGF-β1–induced kidney myofibroblast activation and matrix synthesis is shown.

Techniques Used: Activation Assay

ERK1/2 has a central role in Smad3 and NAD(P)H oxidase signaling of TGF-β1–induced myofibroblast activation. (A) TGF-β1 induced a rapid expression of p-ERK1/2 at 5 min, disappearing thereafter, and was absent up to 24 h after stimulation
Figure Legend Snippet: ERK1/2 has a central role in Smad3 and NAD(P)H oxidase signaling of TGF-β1–induced myofibroblast activation. (A) TGF-β1 induced a rapid expression of p-ERK1/2 at 5 min, disappearing thereafter, and was absent up to 24 h after stimulation

Techniques Used: Activation Assay, Expressing

Related Articles

Synthesized:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Quantitative real-time (qRT) PCR using cDNA (10 ng total RNA equivalent) synthesized using the Proscript cDNA Synthesis Kit (New England Biolabs) was performed in duplicate per sample as described ( ). .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Quantitative RT-PCR:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Paragraph title: qRT-PCR and western blot analysis ... Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Electrophoresis:

Article Title: Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells
Article Snippet: Western Blot Analysis Cell lysates were separated by electrophoresis in a 10% sodium dodecyl sulphate-polyacrylamide gradient minigel (SDS-PAGE) and electrophoretically transferred to a PVDF membrane. .. Western blots were probed with specific antibodies against phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-PTEN, PTEN, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2 (Cell Signaling Technology), GAPDH, PARP, Caspase-3/9, COX-2, p300, NF-κB p50 and p65, CREB-2 and cytochrome c (Santa Cruz Biotech, Santa Cruz, CA).

Incubation:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Following 15 min incubation on ice, cells were sonicated for 20 pulses on ice using a sonic dismembrator (Fisher Scientific) and centrifuged at 14,000 rpm, 4 °C for 5 min to remove cell debris. .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Article Title: Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo
Article Snippet: .. Blots were incubated with the appropriate primary antibodies, including PIK3R2 (1:10000, Abcam), SPRED-1 (1:10000, Abcam), AKT(1:1000, Cell Signaling, Boston, USA), p-AKT (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-ERK1/2 (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech), followed by incubation with HRP-conjugated secondary antibodies and protein visualization by chemiluminescence using ECL Substrate according to the manufacturer’s instructions. ..

Article Title: EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice
Article Snippet: Each nitrocellulose membrane was incubated with specific antibodies. .. Antibodies against p-EGFR Tyr835, EGFR, p-AKT, AKT, p-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Danvers, MA).

Activity Assay:

Article Title: Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy
Article Snippet: Antibodies and Regents The primary antibodies used in this study targeted SDF-1α (Novus Biologicals, Littleton, CO, United States), DPP-4 (Abcam, Cambridge, MA, United States), p-PKA (clone EP2606Y, Abcam, Cambridge, MA, United States), PKA (Cell Signaling, Danvers, MA, United States), NOX2 (clone EPR6991, Abcam, Cambridge, MA, United States), p-ERK1/2 (clone D13.14.4E, Cell Signaling, Danvers, MA, United States), ERK1/2 (Cell Signaling, Danvers, MA, United States), transforming growth factor-β1 (TGF-β1) (clone EPR18163, Abcam, Cambridge, MA, United States), podocin (Abcam, Cambridge, MA, United States), desmin (Abcam, Cambridge, MA, United States), nephrin (clone B-12, Santa Cruz Biotechnology Inc., Dallas, TX, United States), fibronectin (Proteintech, Rosemont, IL, United States), Fsp1 (clone EPR14639(2), Abcam, Cambridge, MA, United States), and α-SMA (clone 1A4, Abcam, Cambridge, MA, United States). .. DPP-4 enzyme activity was tested using a DPP-4 assay kit (Enzo Life Sciences, Farmingdale, NY, United States).

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. To examine whether ERK1/2 can catalyze Ser-177 phosphorylation of SHDAg, we employed a cell-free in vitro kinase assay using highly purified components to examine the enzyme activity on purified SHDAg proteins. ..

Expressing:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Expression level was normalized to Gapdh and E-cadherin ( Cdh1 ; when epithelium-specific genes were examined). .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8). .. This interaction was not observed in experiments using the cells expressing either the S2C or wild-type protein (Fig. , lanes 6 and 7).

BIA-KA:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Protein was quantified via the bicinchoninic acid assay (BCA protein assay kit; Pierce). .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Western Blot:

Article Title: Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells
Article Snippet: .. Western blots were probed with specific antibodies against phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-PTEN, PTEN, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2 (Cell Signaling Technology), GAPDH, PARP, Caspase-3/9, COX-2, p300, NF-κB p50 and p65, CREB-2 and cytochrome c (Santa Cruz Biotech, Santa Cruz, CA). .. The protein bands were detected by enhanced chemiluminescence (Pierce).

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Paragraph title: qRT-PCR and western blot analysis ... Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Paragraph title: Western blotting ... Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Article Title: Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo
Article Snippet: Paragraph title: Western blot analysis ... Blots were incubated with the appropriate primary antibodies, including PIK3R2 (1:10000, Abcam), SPRED-1 (1:10000, Abcam), AKT(1:1000, Cell Signaling, Boston, USA), p-AKT (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-ERK1/2 (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech), followed by incubation with HRP-conjugated secondary antibodies and protein visualization by chemiluminescence using ECL Substrate according to the manufacturer’s instructions.

Article Title: EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice
Article Snippet: Paragraph title: Western Blot Analysis ... Antibodies against p-EGFR Tyr835, EGFR, p-AKT, AKT, p-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Danvers, MA).

Article Title: Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells
Article Snippet: Paragraph title: Western Blot Analysis. ... Primary antibodies for E-cadherin (BD Biosciences), GAPDH, phospho-Akt (Ser473), Akt, phospho-ERK1/2 (Thr202/204), ERK1/2 (Cell Signaling), anti–β-actin antibody (Sigma), α-tubulin (GeneTex), Vimentin (Santa Cruz), and PLCδ1 (Santa Cruz) were used.

Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance
Article Snippet: Paragraph title: Western blot analysis ... The protein extracts were electrophoresed by 12% SDS-PAGE, then electrically transferred onto nitrocellulose filters and probed with the following primary antibodies (specific to the proteins that may associated with acquired drug resistance): YB-1 (dilution factor 1:200, Cell Signalling), c-Kit (1:1000, Cell Signalling); ERK1/2 (1: 1000, Cell Signalling); ERK3 (1: 500, Novus Biologicals); FAS (1: 500, Santa Cruz Biotechnology); MAPT (1: 1500, Proteintech Group); and MDR1 (1: 500, Santa Cruz Biotechnology), ABCB5 (1: 2000, ProSci); PARP-1 (1: 2000, Trevigen); β-Actin (1:5000, Sigma) was used as a loading control.

Article Title: Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor
Article Snippet: Paragraph title: Western blotting ... Antibodies used were the following: ERK1/2 (137F5; 4695; Cell Signaling, The Netherlands), P-ERK1/2 (E-4;SC-7383; Santa Cruz Biotechnology, Texas), JNK1/2 (9252; Cell Signaling), P-JNK1/2 (81E11; 4668; Cell Signaling), P38 (9212; Cell Signaling), P-P38 (9211; Cell Signaling), P-STAT1 (Tyr-701; 9167; Cell Signaling), α-Tubulin (Cedarlane, Canada) and PARP1 (4C10-5, BD Biosciences, The Netherlands).

Kinase Assay:

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. To examine whether ERK1/2 can catalyze Ser-177 phosphorylation of SHDAg, we employed a cell-free in vitro kinase assay using highly purified components to examine the enzyme activity on purified SHDAg proteins. ..

Derivative Assay:

Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance
Article Snippet: The protein extracts were electrophoresed by 12% SDS-PAGE, then electrically transferred onto nitrocellulose filters and probed with the following primary antibodies (specific to the proteins that may associated with acquired drug resistance): YB-1 (dilution factor 1:200, Cell Signalling), c-Kit (1:1000, Cell Signalling); ERK1/2 (1: 1000, Cell Signalling); ERK3 (1: 500, Novus Biologicals); FAS (1: 500, Santa Cruz Biotechnology); MAPT (1: 1500, Proteintech Group); and MDR1 (1: 500, Santa Cruz Biotechnology), ABCB5 (1: 2000, ProSci); PARP-1 (1: 2000, Trevigen); β-Actin (1:5000, Sigma) was used as a loading control. .. The intensity of the band derived from each sample was measured by AlphaImager® HP densitometer.

Protease Inhibitor:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Protein lysates (10 μg), prepared by homogenizing the tissues or cultured cells in RIPA lysis buffer [10 mM Tris-Cl pH 7.2, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 50 mM NaF, 1% sodium deoxycholate, 1 mM PMSF, 1× protease inhibitor mixture set V (EMD Chemicals), 0.2 mM sodium vanadate] were used in western blot analysis. .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Article Title: Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor
Article Snippet: Western blotting For western blotting, cells were resuspended in lysis buffer containing protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche). .. Antibodies used were the following: ERK1/2 (137F5; 4695; Cell Signaling, The Netherlands), P-ERK1/2 (E-4;SC-7383; Santa Cruz Biotechnology, Texas), JNK1/2 (9252; Cell Signaling), P-JNK1/2 (81E11; 4668; Cell Signaling), P38 (9212; Cell Signaling), P-P38 (9211; Cell Signaling), P-STAT1 (Tyr-701; 9167; Cell Signaling), α-Tubulin (Cedarlane, Canada) and PARP1 (4C10-5, BD Biosciences, The Netherlands).

Cell Culture:

Article Title: Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase
Article Snippet: .. To confirm the link between the increased Gαi and enhanced activation of ERK1/2, cultured ASM cells from NTG and AC5 mice were treated with PTX and extracts blotted for total and pERK1/2. ..

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Protein lysates (10 μg), prepared by homogenizing the tissues or cultured cells in RIPA lysis buffer [10 mM Tris-Cl pH 7.2, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 50 mM NaF, 1% sodium deoxycholate, 1 mM PMSF, 1× protease inhibitor mixture set V (EMD Chemicals), 0.2 mM sodium vanadate] were used in western blot analysis. .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Article Title: EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice
Article Snippet: Western Blot Analysis Cell lysate was prepared from cultured cells or homogenized myocardial tissue samples. .. Antibodies against p-EGFR Tyr835, EGFR, p-AKT, AKT, p-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Danvers, MA).

Protein Concentration:

Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance
Article Snippet: Western blot analysis The same numbers of MCF-7 cells (5 × 107 ) were lysed using 150 μl RIPA buffer with freshly added protease inhibitors, then transferred to an Eppendorf microcentrifuge tube and mixed by inversion for 30 m at 4°C and centrifuged at 14000 rpm for 30 m. The supernatant was transferred to a new Eppendorf tube, the protein concentration was determined by the Bradford method. .. The protein extracts were electrophoresed by 12% SDS-PAGE, then electrically transferred onto nitrocellulose filters and probed with the following primary antibodies (specific to the proteins that may associated with acquired drug resistance): YB-1 (dilution factor 1:200, Cell Signalling), c-Kit (1:1000, Cell Signalling); ERK1/2 (1: 1000, Cell Signalling); ERK3 (1: 500, Novus Biologicals); FAS (1: 500, Santa Cruz Biotechnology); MAPT (1: 1500, Proteintech Group); and MDR1 (1: 500, Santa Cruz Biotechnology), ABCB5 (1: 2000, ProSci); PARP-1 (1: 2000, Trevigen); β-Actin (1:5000, Sigma) was used as a loading control.

Polymerase Chain Reaction:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: PCR primers are listed in Table S1 and as described previously ( ). .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Sonication:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Following 15 min incubation on ice, cells were sonicated for 20 pulses on ice using a sonic dismembrator (Fisher Scientific) and centrifuged at 14,000 rpm, 4 °C for 5 min to remove cell debris. .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Mutagenesis:

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8). .. This interaction was not observed in experiments using the cells expressing either the S2C or wild-type protein (Fig. , lanes 6 and 7).

Isolation:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Total RNA was isolated at least in triplicate from NP/MxP tissues freshly dissected from embryos and from cultured NP/MxP explants using Trizol (Sigma). .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Mouse Assay:

Article Title: Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase
Article Snippet: .. To confirm the link between the increased Gαi and enhanced activation of ERK1/2, cultured ASM cells from NTG and AC5 mice were treated with PTX and extracts blotted for total and pERK1/2. ..

Purification:

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. To examine whether ERK1/2 can catalyze Ser-177 phosphorylation of SHDAg, we employed a cell-free in vitro kinase assay using highly purified components to examine the enzyme activity on purified SHDAg proteins. ..

SDS Page:

Article Title: Ursolic Acid Simultaneously Targets Multiple Signaling Pathways to Suppress Proliferation and Induce Apoptosis in Colon Cancer Cells
Article Snippet: Western Blot Analysis Cell lysates were separated by electrophoresis in a 10% sodium dodecyl sulphate-polyacrylamide gradient minigel (SDS-PAGE) and electrophoretically transferred to a PVDF membrane. .. Western blots were probed with specific antibodies against phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-mTOR, mTOR, phospho-PTEN, PTEN, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2 (Cell Signaling Technology), GAPDH, PARP, Caspase-3/9, COX-2, p300, NF-κB p50 and p65, CREB-2 and cytochrome c (Santa Cruz Biotech, Santa Cruz, CA).

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma). .. Primary antibodies were detected using HRP-conjugated anti-rabbit or anti-mouse IgG and visualized by SuperSignal West chemiluminescent substrate (Pierce).

Article Title: Upregulation of miR-126-3p promotes human saphenous vein endothelial cell proliferation in vitro and prevents vein graft neointimal formation ex vivo and in vivo
Article Snippet: Equal amounts of proteins were resolved by SDS-PAGE and transferred to PVDF membranes. .. Blots were incubated with the appropriate primary antibodies, including PIK3R2 (1:10000, Abcam), SPRED-1 (1:10000, Abcam), AKT(1:1000, Cell Signaling, Boston, USA), p-AKT (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-ERK1/2 (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech), followed by incubation with HRP-conjugated secondary antibodies and protein visualization by chemiluminescence using ECL Substrate according to the manufacturer’s instructions.

Article Title: EGFR Inhibition Blocks Palmitic Acid-induced inflammation in cardiomyocytes and Prevents Hyperlipidemia-induced Cardiac Injury in Mice
Article Snippet: 50–90 mg of lysates were separated by 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. .. Antibodies against p-EGFR Tyr835, EGFR, p-AKT, AKT, p-ERK1/2, and ERK1/2 were from Cell Signaling Technology (Danvers, MA).

Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance
Article Snippet: .. The protein extracts were electrophoresed by 12% SDS-PAGE, then electrically transferred onto nitrocellulose filters and probed with the following primary antibodies (specific to the proteins that may associated with acquired drug resistance): YB-1 (dilution factor 1:200, Cell Signalling), c-Kit (1:1000, Cell Signalling); ERK1/2 (1: 1000, Cell Signalling); ERK3 (1: 500, Novus Biologicals); FAS (1: 500, Santa Cruz Biotechnology); MAPT (1: 1500, Proteintech Group); and MDR1 (1: 500, Santa Cruz Biotechnology), ABCB5 (1: 2000, ProSci); PARP-1 (1: 2000, Trevigen); β-Actin (1:5000, Sigma) was used as a loading control. .. The membranes were rinsed with Tris-buffered saline, treated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) was applied for 1 h at 1:2000 dilution.

Software:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology). .. The signals were quantified using ImageJ software, and each experiment was performed in triplicate.

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma). .. Band densities were evaluated using ImageJ software, and normalized to total levels of respective proteins or to β-actin.

DCFH-DA Assay:

Article Title: Saxagliptin Attenuates Albuminuria by Inhibiting Podocyte Epithelial- to-Mesenchymal Transition via SDF-1α in Diabetic Nephropathy
Article Snippet: Antibodies and Regents The primary antibodies used in this study targeted SDF-1α (Novus Biologicals, Littleton, CO, United States), DPP-4 (Abcam, Cambridge, MA, United States), p-PKA (clone EP2606Y, Abcam, Cambridge, MA, United States), PKA (Cell Signaling, Danvers, MA, United States), NOX2 (clone EPR6991, Abcam, Cambridge, MA, United States), p-ERK1/2 (clone D13.14.4E, Cell Signaling, Danvers, MA, United States), ERK1/2 (Cell Signaling, Danvers, MA, United States), transforming growth factor-β1 (TGF-β1) (clone EPR18163, Abcam, Cambridge, MA, United States), podocin (Abcam, Cambridge, MA, United States), desmin (Abcam, Cambridge, MA, United States), nephrin (clone B-12, Santa Cruz Biotechnology Inc., Dallas, TX, United States), fibronectin (Proteintech, Rosemont, IL, United States), Fsp1 (clone EPR14639(2), Abcam, Cambridge, MA, United States), and α-SMA (clone 1A4, Abcam, Cambridge, MA, United States). .. ROS levels in renal tissue were tested with a dihydroethidium (DHE) assay kit (Beyotime, Jiangsu, China), and ROS levels in cells were examined with a DCFH-DA assay kit (Beyotime, Jiangsu, China).

In Vitro:

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. To examine whether ERK1/2 can catalyze Ser-177 phosphorylation of SHDAg, we employed a cell-free in vitro kinase assay using highly purified components to examine the enzyme activity on purified SHDAg proteins. ..

Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿
Article Snippet: .. These data strongly suggest that ERK1/2 is sufficient to directly phosphorylate SHDAg at the Ser-177 residue in vitro. .. Taken together, our results indicate that ERK1/2 are capable of catalyzing phosphorylation of SHDAg at Ser-177 both in vivo and in vitro.

Knock-Out:

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells
Article Snippet: .. These effects are consistent with past findings of a Schwann cell-specific knockout of Erk1/2 ( ) which showed increased Mbp and Mag levels in E12.5 DRG samples even when Egr2 levels were repressed. .. The role of Erk1/2 signaling in Schwann cell differentiation has been the subject of several studies (reviewed in , ).

Acid Assay:

Article Title: Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1
Article Snippet: Protein was quantified via the bicinchoninic acid assay (BCA protein assay kit; Pierce). .. Aliquots containing 15 μg protein were mixed with 2× reducing sample buffer (containing 4% SDS, 20% glycerol, and 10% β-mercaptoethanol), and separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted for specific proteins with the following primary antibodies: TrxR1: (1:1000, Abcam), Trx1 (1:2000, Cell Signaling), phospho SAPK/JNK1/2 (1:1000, Cell Signaling), SAPK/JNK1/2 (1:1000, Cell Signaling), phospho ERK1/2 (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), phospho P-38 (1:1000, Cell Signaling), P-38 (1:1000, Cell Signaling), HO-1 (1:250, Biovision), NQO1 (1:1000, Cell Signaling), β-actin (1:5000, Sigma).

Activation Assay:

Article Title: Paradoxical attenuation of ?2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase
Article Snippet: .. To confirm the link between the increased Gαi and enhanced activation of ERK1/2, cultured ASM cells from NTG and AC5 mice were treated with PTX and extracts blotted for total and pERK1/2. ..

Article Title: Estrogen modulation of the ethanol-evoked myocardial oxidative stress and dysfunction via DAPK3/Akt/ERK activation in male rats
Article Snippet: .. These events include the increase in ROS generation and lipid peroxidation and phosphorylation (activation) of pro-oxidant and apoptotic proteins, such as DAPK3, Akt, and ERK1/2, in the myocardium. ..

Lysis:

Article Title: Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development
Article Snippet: Protein lysates (10 μg), prepared by homogenizing the tissues or cultured cells in RIPA lysis buffer [10 mM Tris-Cl pH 7.2, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 50 mM NaF, 1% sodium deoxycholate, 1 mM PMSF, 1× protease inhibitor mixture set V (EMD Chemicals), 0.2 mM sodium vanadate] were used in western blot analysis. .. Primary antibodies (all 1:1000 dilution) were against phospho-LRP6 (Cell Signaling), LRP6 (Santa Cruz Biotechnology), phospho-c-JUN (Cell Signaling), phospho-JNK1/2 (Cell Signaling), JNK1/2 (Cell Signaling), phospho-ROCK2 (Abcam), ROCK2 (Santa Cruz Biotechnology), phospho-ERK1/2 (Cell Signaling), ERK1/2 (Cell Signaling) and α-tubulin (Santa Cruz Biotechnology).

Article Title: Lithocholic acid controls adaptive immune responses by inhibition of Th1 activation through the Vitamin D receptor
Article Snippet: Western blotting For western blotting, cells were resuspended in lysis buffer containing protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche). .. Antibodies used were the following: ERK1/2 (137F5; 4695; Cell Signaling, The Netherlands), P-ERK1/2 (E-4;SC-7383; Santa Cruz Biotechnology, Texas), JNK1/2 (9252; Cell Signaling), P-JNK1/2 (81E11; 4668; Cell Signaling), P38 (9212; Cell Signaling), P-P38 (9211; Cell Signaling), P-STAT1 (Tyr-701; 9167; Cell Signaling), α-Tubulin (Cedarlane, Canada) and PARP1 (4C10-5, BD Biosciences, The Netherlands).

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    Cell Signaling Technology Inc erk1 2 antibody
    (A) Immunoblot analysis of <t>ERK1/2</t> from the pulled down fractions obtained after competition experiments between SCH-TCO and a range of concentrations of selected ERK1/2 inhibitors in HCT116 cells. (B) Graphs showing the target occupancies of LY3214996, GDC-0994 and SCH772984 determined in competition mode with SCH-TCO in HCT116 cells.
    Erk1 2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic presentation of proposed biglycan/LRP6/IGF-IR downstream signaling axis in regulation of MG63 osteosarcoma cell growth. (A) Secreted to ECM biglycan binds to LRP6 and activates frizzled to de-activate GSK3β. (B) β-catenin cytoplasmic pool increases. (C) β-catenin co-localizes with IGF-IR to enhance its activation/deposition to membrane and to (D) cytoplasm. (E) pIGF-IR activates <t>ERK1,2</t> to induce downstream transcriptional regulation. (F) Part of cytoplasmic β-catenin pool translocates to nucleus to induce transcriptional regulation of target genes.
    Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Goniolactone C inhibits the <t>PDGFR-β/ERK1/2</t> cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).
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    (A) Immunoblot analysis of ERK1/2 from the pulled down fractions obtained after competition experiments between SCH-TCO and a range of concentrations of selected ERK1/2 inhibitors in HCT116 cells. (B) Graphs showing the target occupancies of LY3214996, GDC-0994 and SCH772984 determined in competition mode with SCH-TCO in HCT116 cells.

    Journal: Chemical Science

    Article Title: Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02754d

    doi: 10.1039/c8sc02754d

    Figure Lengend Snippet: (A) Immunoblot analysis of ERK1/2 from the pulled down fractions obtained after competition experiments between SCH-TCO and a range of concentrations of selected ERK1/2 inhibitors in HCT116 cells. (B) Graphs showing the target occupancies of LY3214996, GDC-0994 and SCH772984 determined in competition mode with SCH-TCO in HCT116 cells.

    Article Snippet: The ERK1/2 antibody was bought from Cell Signaling Technology (9102S).

    Techniques:

    (A) Examples of ERK1/2 inhibitors classified in two categories: Type 1 and pERK modulating inhibitors. (B) Co-crystal structure of SCH772984 with ERK2 (pdb: 4QTA ). 21 The inhibitor binds to an altered conformation of ERK2 allowing interaction with the Tyr64 side chain. The two sites identified to extend the molecule and insert the TCO tag are highlighted (red arrows). The structural design of the two probes, TCO-SCH and SCH-TCO are shown. (C) Co-crystal structure of GDC-0994 with ERK2 (pdb:; 5K4I ) 17 showing the vector for the extension of the molecule (red arrow) and structural design of the two probes, TCO-GDC-1 and TCO-GDC-2.

    Journal: Chemical Science

    Article Title: Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02754d

    doi: 10.1039/c8sc02754d

    Figure Lengend Snippet: (A) Examples of ERK1/2 inhibitors classified in two categories: Type 1 and pERK modulating inhibitors. (B) Co-crystal structure of SCH772984 with ERK2 (pdb: 4QTA ). 21 The inhibitor binds to an altered conformation of ERK2 allowing interaction with the Tyr64 side chain. The two sites identified to extend the molecule and insert the TCO tag are highlighted (red arrows). The structural design of the two probes, TCO-SCH and SCH-TCO are shown. (C) Co-crystal structure of GDC-0994 with ERK2 (pdb:; 5K4I ) 17 showing the vector for the extension of the molecule (red arrow) and structural design of the two probes, TCO-GDC-1 and TCO-GDC-2.

    Article Snippet: The ERK1/2 antibody was bought from Cell Signaling Technology (9102S).

    Techniques: Plasmid Preparation

    Genotypic comparisons of ERK1/2 and CREB expressions in the amygdala of naïve WT and nNOS KO mice (n=9–14/group). No differences in ERK1/2 phosphorylation or expression were observed, however pCREB was significantly elevated in nNOS KO mice compared to WT mice. (A) Representative immunoblots for ERK1/2 experiments. (B) No genotypic differences were observed for pERK1/2. (C) No genotypic differences were observed for ERK1/2 expression. (D) Representative immunoblots for CREB experiments. (E) pCREB levels were 2-fold higher in nNOS KO mice than in WT mice (* P =0.005). (F) No genotypic differences were observed for total CREB expression.

    Journal: Neuroscience

    Article Title: Long-term memory of visually cued fear conditioning: roles of the nNOS gene and CREB

    doi: 10.1016/j.neuroscience.2010.11.005

    Figure Lengend Snippet: Genotypic comparisons of ERK1/2 and CREB expressions in the amygdala of naïve WT and nNOS KO mice (n=9–14/group). No differences in ERK1/2 phosphorylation or expression were observed, however pCREB was significantly elevated in nNOS KO mice compared to WT mice. (A) Representative immunoblots for ERK1/2 experiments. (B) No genotypic differences were observed for pERK1/2. (C) No genotypic differences were observed for ERK1/2 expression. (D) Representative immunoblots for CREB experiments. (E) pCREB levels were 2-fold higher in nNOS KO mice than in WT mice (* P =0.005). (F) No genotypic differences were observed for total CREB expression.

    Article Snippet: Following development of the immunoblots for the phosphorylated protein, the immunoblots were stripped with Restore western blot stripping buffer (Pierce) for 15 min. After 3 quick rinses in TBS-T, the membranes were blocked again as above and then incubated with anti-total CREB (1:1000) or anti-total ERK1/2 (1:3000) antibodies (both Cell Signaling) for 1h at RT in TBS-T + 5% BSA.

    Techniques: Mouse Assay, Expressing, Western Blot

    Schematic presentation of proposed biglycan/LRP6/IGF-IR downstream signaling axis in regulation of MG63 osteosarcoma cell growth. (A) Secreted to ECM biglycan binds to LRP6 and activates frizzled to de-activate GSK3β. (B) β-catenin cytoplasmic pool increases. (C) β-catenin co-localizes with IGF-IR to enhance its activation/deposition to membrane and to (D) cytoplasm. (E) pIGF-IR activates ERK1,2 to induce downstream transcriptional regulation. (F) Part of cytoplasmic β-catenin pool translocates to nucleus to induce transcriptional regulation of target genes.

    Journal: Frontiers in Oncology

    Article Title: Biglycan Regulates MG63 Osteosarcoma Cell Growth Through a LPR6/β-Catenin/IGFR-IR Signaling Axis

    doi: 10.3389/fonc.2018.00470

    Figure Lengend Snippet: Schematic presentation of proposed biglycan/LRP6/IGF-IR downstream signaling axis in regulation of MG63 osteosarcoma cell growth. (A) Secreted to ECM biglycan binds to LRP6 and activates frizzled to de-activate GSK3β. (B) β-catenin cytoplasmic pool increases. (C) β-catenin co-localizes with IGF-IR to enhance its activation/deposition to membrane and to (D) cytoplasm. (E) pIGF-IR activates ERK1,2 to induce downstream transcriptional regulation. (F) Part of cytoplasmic β-catenin pool translocates to nucleus to induce transcriptional regulation of target genes.

    Article Snippet: These results suggest that ERK1/2 is a downstream mediator of IGF-IR/biglycan signaling dependent cell growth.

    Techniques: Activation Assay

    The role ofERK1/2 on MG63 cell proliferation and the effect of biglycan expression on its activation. (A) MG63 cells were harvested and seeded (3,500 cells/well) on 96-well plates. Cells, in each well, incubated with serum-free medium (control), 10 ng/ml IGF-I (IGF-I), 5 μM ERK1/2 inhibitor (InhERK1/2), and 10 ng/ml IGF-I + 5 μM ERK1/2 inhibitor (IGF-I+InhERK1/2), were counted using fluorometric CyQUANT assay kit. (B) Expression of ERK1/2 total protein (ERK1/2) and phosphorylated ERK1/2 protein (pERK1/2) of cells incubated in serum-free medium (control) and cells transfected with either siRNAs against biglycan (siBGN) or scrambled siRNAs (siScr) were determined by Western blot analysis. (C) Densitometric analysis of the activated ERK1/2 levels (pERK1/2 /ERK1/2) from the proteinbandswere normalized against actin and plotted. Results represent the average of three separate experiments. Means ± S.E.M were plotted; statistical significance: *** p ≤ 0.001, ** p ≤ 0.01 compared with the respective control samples.

    Journal: Frontiers in Oncology

    Article Title: Biglycan Regulates MG63 Osteosarcoma Cell Growth Through a LPR6/β-Catenin/IGFR-IR Signaling Axis

    doi: 10.3389/fonc.2018.00470

    Figure Lengend Snippet: The role ofERK1/2 on MG63 cell proliferation and the effect of biglycan expression on its activation. (A) MG63 cells were harvested and seeded (3,500 cells/well) on 96-well plates. Cells, in each well, incubated with serum-free medium (control), 10 ng/ml IGF-I (IGF-I), 5 μM ERK1/2 inhibitor (InhERK1/2), and 10 ng/ml IGF-I + 5 μM ERK1/2 inhibitor (IGF-I+InhERK1/2), were counted using fluorometric CyQUANT assay kit. (B) Expression of ERK1/2 total protein (ERK1/2) and phosphorylated ERK1/2 protein (pERK1/2) of cells incubated in serum-free medium (control) and cells transfected with either siRNAs against biglycan (siBGN) or scrambled siRNAs (siScr) were determined by Western blot analysis. (C) Densitometric analysis of the activated ERK1/2 levels (pERK1/2 /ERK1/2) from the proteinbandswere normalized against actin and plotted. Results represent the average of three separate experiments. Means ± S.E.M were plotted; statistical significance: *** p ≤ 0.001, ** p ≤ 0.01 compared with the respective control samples.

    Article Snippet: These results suggest that ERK1/2 is a downstream mediator of IGF-IR/biglycan signaling dependent cell growth.

    Techniques: Expressing, Activation Assay, Incubation, CyQUANT Assay, Transfection, Western Blot

    Goniolactone C inhibits the PDGFR-β/ERK1/2 cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).

    Journal: Molecules

    Article Title: Goniolactone C, a Styryl Lactone Derivative, Inhibits PDGF-BB-Induced Vascular Smooth Muscle Cell Migration and Proliferation via PDGFR/ERK Signaling

    doi: 10.3390/molecules191219501

    Figure Lengend Snippet: Goniolactone C inhibits the PDGFR-β/ERK1/2 cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).

    Article Snippet: Goniolactone C Inhibits the PDGFR-β/ERK1/2 Cell Signaling Cascade PDGF-BB activates PDGFR-β on VSMCs, is one of the most potent mitogens and chemoattractants for vascular smooth muscle cells (VSMCs), and plays a central role in the onset and development of various vascular disorders [ , , ].

    Techniques: In Vitro, Activity Assay, Incubation, Expressing, SDS Page, Western Blot