Structured Review

Cell Signaling Technology Inc phospho erk1 2
VUN100b binds and inhibits US28 signaling. A) ELISA binding of monovalent VUN100 and bivalent VUN100b to membrane extracts of US28-expressing HEK293T cells. B) Displacement of 125 I-CX3CL1 from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and VUN100b. C) Effect of nanobodies on US28-mediated NFAT (Nuclear Factor of Activated T-cells) activation. HEK293T cells expressing US28 and containing an NFAT-luciferase reporter were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 24 h prior to luminescence measurement. D) Immunofluorescence microscopy of nanobody binding to US28-expressing THP-1 cells. US28 was detected using a polyclonal rabbit anti-US28 antibody (US28 mAb). Bound nanobody was detected using the Myc-tag present on the nanobodies and an anti-Myc antibody (Nb). E) Western blot detection for <t>phospho-ERK1/2</t> of lysates of THP-1 mock transduced cells or US28-expressing THP-1 cells. Cells were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 48 h. Phosphorylated protein levels over total protein levels were determined and normalized to actin protein levels. Relative phosphorylated protein levels were normalized to untreated THP-1 mock cell lysates. Data is plotted as mean ± S.D.. Statistical analyses were performed using unpaired two-tailed t-test. *, p
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1) Product Images from "Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies"

Article Title: Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies

Journal: bioRxiv

doi: 10.1101/2020.05.12.071860

VUN100b binds and inhibits US28 signaling. A) ELISA binding of monovalent VUN100 and bivalent VUN100b to membrane extracts of US28-expressing HEK293T cells. B) Displacement of 125 I-CX3CL1 from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and VUN100b. C) Effect of nanobodies on US28-mediated NFAT (Nuclear Factor of Activated T-cells) activation. HEK293T cells expressing US28 and containing an NFAT-luciferase reporter were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 24 h prior to luminescence measurement. D) Immunofluorescence microscopy of nanobody binding to US28-expressing THP-1 cells. US28 was detected using a polyclonal rabbit anti-US28 antibody (US28 mAb). Bound nanobody was detected using the Myc-tag present on the nanobodies and an anti-Myc antibody (Nb). E) Western blot detection for phospho-ERK1/2 of lysates of THP-1 mock transduced cells or US28-expressing THP-1 cells. Cells were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 48 h. Phosphorylated protein levels over total protein levels were determined and normalized to actin protein levels. Relative phosphorylated protein levels were normalized to untreated THP-1 mock cell lysates. Data is plotted as mean ± S.D.. Statistical analyses were performed using unpaired two-tailed t-test. *, p
Figure Legend Snippet: VUN100b binds and inhibits US28 signaling. A) ELISA binding of monovalent VUN100 and bivalent VUN100b to membrane extracts of US28-expressing HEK293T cells. B) Displacement of 125 I-CX3CL1 from US28-expressing membranes by unlabeled ligand or the nanobodies VUN100 and VUN100b. C) Effect of nanobodies on US28-mediated NFAT (Nuclear Factor of Activated T-cells) activation. HEK293T cells expressing US28 and containing an NFAT-luciferase reporter were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 24 h prior to luminescence measurement. D) Immunofluorescence microscopy of nanobody binding to US28-expressing THP-1 cells. US28 was detected using a polyclonal rabbit anti-US28 antibody (US28 mAb). Bound nanobody was detected using the Myc-tag present on the nanobodies and an anti-Myc antibody (Nb). E) Western blot detection for phospho-ERK1/2 of lysates of THP-1 mock transduced cells or US28-expressing THP-1 cells. Cells were untreated (untr) or treated with an irrelevant nanobody (Irr Nb), VUN100 or VUN100b for 48 h. Phosphorylated protein levels over total protein levels were determined and normalized to actin protein levels. Relative phosphorylated protein levels were normalized to untreated THP-1 mock cell lysates. Data is plotted as mean ± S.D.. Statistical analyses were performed using unpaired two-tailed t-test. *, p

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Expressing, Activation Assay, Luciferase, Immunofluorescence, Microscopy, Western Blot, Two Tailed Test

2) Product Images from "Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization"

Article Title: Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201709137

SubAB, but not other AB 5 toxins, cleaves BiP and induces S729 phosphorylation of IRE1. (A) 5TGM1 cells were treated with mutant or native SubAB for 24 h using increasing concentrations and immunoblotted. (B) 5TGM1 cells were treated with 1 nM SubAB, 1 nM Shiga toxin 1, or 1 nM Shiga toxin 2 for 24 h and immunoblotted. Phospho–NF-κB p65 served as a control for the activity of the Shiga toxins. (C) 5TGM1 cells were treated with 1 nM SubAB for 24 h or with 1 nM cholera toxin for 24 h and immunoblotted. Phospho-ERK1/2 and phospho–NF-κB p105 served as controls for the activity of the cholera toxin. (D) 5TGM1 cells were treated with 1 nM SubAB or 1 nM pertussis toxin for 24 h and immunoblotted. Phospho–NF-κB p65 served as a control for pertussis toxin activity. Data in this figure are representative of three independent experiments.
Figure Legend Snippet: SubAB, but not other AB 5 toxins, cleaves BiP and induces S729 phosphorylation of IRE1. (A) 5TGM1 cells were treated with mutant or native SubAB for 24 h using increasing concentrations and immunoblotted. (B) 5TGM1 cells were treated with 1 nM SubAB, 1 nM Shiga toxin 1, or 1 nM Shiga toxin 2 for 24 h and immunoblotted. Phospho–NF-κB p65 served as a control for the activity of the Shiga toxins. (C) 5TGM1 cells were treated with 1 nM SubAB for 24 h or with 1 nM cholera toxin for 24 h and immunoblotted. Phospho-ERK1/2 and phospho–NF-κB p105 served as controls for the activity of the cholera toxin. (D) 5TGM1 cells were treated with 1 nM SubAB or 1 nM pertussis toxin for 24 h and immunoblotted. Phospho–NF-κB p65 served as a control for pertussis toxin activity. Data in this figure are representative of three independent experiments.

Techniques Used: Mutagenesis, Activity Assay

3) Product Images from "hMAGEA2 promotes progression of breast cancer by regulating Akt and Erk1/2 pathways"

Article Title: hMAGEA2 promotes progression of breast cancer by regulating Akt and Erk1/2 pathways

Journal: Oncotarget

doi: 10.18632/oncotarget.16184

hMAGEA2 activates p-Akt and p-Erk1/2 in TNBC cell line ( A ) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-JNK, total-JNK, phospho-p38, and total-p38, measured in MDA-MB-231 cell line overexpressing hMAGEA2 and those not overexpressing hMAGEA2. β-actin was used as loading control. ( B ) Relative protein expression level of p-Akt and p-Erk1/2 in MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2. (Means ± SD, * p
Figure Legend Snippet: hMAGEA2 activates p-Akt and p-Erk1/2 in TNBC cell line ( A ) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-JNK, total-JNK, phospho-p38, and total-p38, measured in MDA-MB-231 cell line overexpressing hMAGEA2 and those not overexpressing hMAGEA2. β-actin was used as loading control. ( B ) Relative protein expression level of p-Akt and p-Erk1/2 in MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2. (Means ± SD, * p

Techniques Used: Expressing, Multiple Displacement Amplification

Knockdown of hMAGEA2 inhibits progression of TNBC ( A ) Establishment of hMAGEA2 knockdown in MDA-MB-468 cell line. Decrease in the expression of hMAGEA2 mRNA (left panel) and protein (right panel) compared with those of scrambled control shRNA and shMAGEA2. ( B ) Relative OD values were measured at 0, 12, 24, 36, and 48 h in scrambled control shRNA and shMAGEA2 using the CCK-8 assay (upper panel). Colony formation was compared between scrambled control shRNA and shMAGEA2 (x50 magnification, scale bar = 250 μm) (lower panel). ( C ) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38 was assessed in scrambled control shRNA and shMAGEA2. ( D ) Growth curve of xenograft tumor formation after injection of cell lines expressing shMAGEA2 and scrambled control shRNA in Balb/c nude mice (left panel). Using sectioned xenograft tumor tissues, H E staining and immunohistochemistry were performed. (x50 magnification, scale bar = 250 μm) (right panel). (Means ± SD, * p
Figure Legend Snippet: Knockdown of hMAGEA2 inhibits progression of TNBC ( A ) Establishment of hMAGEA2 knockdown in MDA-MB-468 cell line. Decrease in the expression of hMAGEA2 mRNA (left panel) and protein (right panel) compared with those of scrambled control shRNA and shMAGEA2. ( B ) Relative OD values were measured at 0, 12, 24, 36, and 48 h in scrambled control shRNA and shMAGEA2 using the CCK-8 assay (upper panel). Colony formation was compared between scrambled control shRNA and shMAGEA2 (x50 magnification, scale bar = 250 μm) (lower panel). ( C ) Protein expression of phospho-Akt, total-Akt, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38 was assessed in scrambled control shRNA and shMAGEA2. ( D ) Growth curve of xenograft tumor formation after injection of cell lines expressing shMAGEA2 and scrambled control shRNA in Balb/c nude mice (left panel). Using sectioned xenograft tumor tissues, H E staining and immunohistochemistry were performed. (x50 magnification, scale bar = 250 μm) (right panel). (Means ± SD, * p

Techniques Used: Multiple Displacement Amplification, Expressing, shRNA, CCK-8 Assay, Injection, Mouse Assay, Staining, Immunohistochemistry

hMAGEA2 enhances xenograft tumor formation via activation of p-Akt and p-Erk1/2 ( A ) Growth curve of xenograft tumor formation after the injection of MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2, in Balb/c nude mice. (Means ± SD, * p
Figure Legend Snippet: hMAGEA2 enhances xenograft tumor formation via activation of p-Akt and p-Erk1/2 ( A ) Growth curve of xenograft tumor formation after the injection of MDA-MB-231 cell line overexpressing hMAGEA2, and those not overexpressing hMAGEA2, in Balb/c nude mice. (Means ± SD, * p

Techniques Used: Activation Assay, Injection, Multiple Displacement Amplification, Mouse Assay

4) Product Images from "The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage"

Article Title: The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.381

AKT and ERK1/2 contribute differently to the neuroprotective effects of MK-X and EPO. ( a ) MK-X- and EPO-induced Erk1/2 and Akt activation in the presence or absence of U0126 (1 h) under oxidative stress condition. MK-X and EPO increased pErk1/2 levels. These effects were completely blocked by pre-incubation with U0126. ( b ) Quantitative analysis Erk1/2 and Akt activation in five independent experiments. ( c ) MK-X- and EPO-induced Akt activation in the presence or absence of LY294002. MK-X and EPO increased pAkt levels. These effects were completely blocked by pre-incubation with LY294002. ( d ) Quantitative analysis of Akt activation in five independent experiments. ( e ) Neuroprotective effects of treatments with specific inhibitors. In the presence of 30 μ M glutamate, cells were incubated with EPO (1 IU) or MK-X (1 pM). A culture not treated with glutamate was used as a positive control. To block the activation of ERK1/2 and Akt, cells were incubated with 20 μ M U0126 and50 μ M LY294002, respectively. Cell viability was assessed by the Calcein-AM assay. Calcium accumulation ( f ), TMRM staining ( g ), and ROS generation ( h ) were observed in cells treated with EPO (1 IU) or MK-X (1 pM) and a specific inhibitor (20 μ M U0126 or 50 μ M LY294002). ( i ) Immunoblots for Bcl-2 and GAPDH after incubation for 12 h with EPO (1 IU) or MK-X (1 pM) and a specific inhibitor (20 μ M U0126 or 50 μ M LY294002). ( j ) Protein levels were quantified by stereological analysis using the ImageJ program. Equal loading was confirmed by monitoring the GAPDH protein level. Data are means±S.D. from five independent experiments. For statistical analysis, one-way ANOVA was performed, followed by Dunnett’s post hoc test. Statistical significance is denoted (* P
Figure Legend Snippet: AKT and ERK1/2 contribute differently to the neuroprotective effects of MK-X and EPO. ( a ) MK-X- and EPO-induced Erk1/2 and Akt activation in the presence or absence of U0126 (1 h) under oxidative stress condition. MK-X and EPO increased pErk1/2 levels. These effects were completely blocked by pre-incubation with U0126. ( b ) Quantitative analysis Erk1/2 and Akt activation in five independent experiments. ( c ) MK-X- and EPO-induced Akt activation in the presence or absence of LY294002. MK-X and EPO increased pAkt levels. These effects were completely blocked by pre-incubation with LY294002. ( d ) Quantitative analysis of Akt activation in five independent experiments. ( e ) Neuroprotective effects of treatments with specific inhibitors. In the presence of 30 μ M glutamate, cells were incubated with EPO (1 IU) or MK-X (1 pM). A culture not treated with glutamate was used as a positive control. To block the activation of ERK1/2 and Akt, cells were incubated with 20 μ M U0126 and50 μ M LY294002, respectively. Cell viability was assessed by the Calcein-AM assay. Calcium accumulation ( f ), TMRM staining ( g ), and ROS generation ( h ) were observed in cells treated with EPO (1 IU) or MK-X (1 pM) and a specific inhibitor (20 μ M U0126 or 50 μ M LY294002). ( i ) Immunoblots for Bcl-2 and GAPDH after incubation for 12 h with EPO (1 IU) or MK-X (1 pM) and a specific inhibitor (20 μ M U0126 or 50 μ M LY294002). ( j ) Protein levels were quantified by stereological analysis using the ImageJ program. Equal loading was confirmed by monitoring the GAPDH protein level. Data are means±S.D. from five independent experiments. For statistical analysis, one-way ANOVA was performed, followed by Dunnett’s post hoc test. Statistical significance is denoted (* P

Techniques Used: Activation Assay, Incubation, Positive Control, Blocking Assay, Calcein AM Assay, Staining, Western Blot

5) Product Images from "Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation"

Article Title: Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.409

THOC5 is required for the processing of Ets1 . ( a ) Human c-Fms cDNA was expressed in MEF (M-CSFR THOC5 (flox/flox) MEF) cells. After serum starvation for 24 h, cells were stimulated with human M-CSF (100 ng/ml) for 0.5, 2 and 4 h and cell extracts were then subjected to Fms-, phosphoErk (p-Erk1/2)-, Erk-, phosphoAkt (p-Akt)-, Akt-, and GAPDH-specific immunoblot. ( b ) M-CSFR (flox/flox) MEF cells were treated as described in ( a ) and were stimulated with M-CSF for 1, 2 and 4 h. Total RNAs were isolated and subjected to Ets1 -, Ets2 -, Etv5 -, Egr1 -, Id1 -, Id3 -, HoxA1 - and GAPDH -specific reverse transcriptase (RT)-PCR. ( c ) Sister cultures of M-CSFR THOC5 (flox/flox) MEF were infected with Ad-GFP or Ad-GFP-Cre virus for 3 days. After serum starvation for 24 h, cells were stimulated with M-CSF for 1 and 2 h. Protein extract from cells before stimulation (0 h) were supplied for THOC5- and GAPDH-specific immunoblot (Blot). Total RNAs were isolated from each preparation and supplied for Ets1 -, Ets2 -, Egr1 -, GAPDH - and THOC5 -specific RT-PCR (RT-PCR). ( d ) Aliquots of cDNA samples from ( c ) were applied for quantitative RT-PCR analysis of Ets1 mRNA (TaqMan RT-PCR). Relative expression levels compared with GAPDH were normalized. Average values from four independent PCR reactions+S.D. are shown. ( e ) Cells were prepared as described in ( c ), but RNA and protein were isolated from the nuclear and the cytoplasmic fractions obtained from 5000 cells and then supplied for Ets1 -, Ets2 -, Egr1 -, and GAPDH -specific RT-PCR. Protein extracts were supplied for GAPDH- and Histone H3-specific immunoblot. ( f ) RNA was isolated from the nuclear fraction as described in ( a ) and subjected to Ets1 - and GAPDH -specific RT-PCR using an exon–intron primer pair as indicated. RNA samples were reverse transcribed using oligo dT or random hexamer (random) primer as indicated ( Supplementary Table S1 ). As negative controls, RNA samples were supplied for PCR without reverse transcription (−). We performed independent experiments, and we show one example of representative data ( a–f )
Figure Legend Snippet: THOC5 is required for the processing of Ets1 . ( a ) Human c-Fms cDNA was expressed in MEF (M-CSFR THOC5 (flox/flox) MEF) cells. After serum starvation for 24 h, cells were stimulated with human M-CSF (100 ng/ml) for 0.5, 2 and 4 h and cell extracts were then subjected to Fms-, phosphoErk (p-Erk1/2)-, Erk-, phosphoAkt (p-Akt)-, Akt-, and GAPDH-specific immunoblot. ( b ) M-CSFR (flox/flox) MEF cells were treated as described in ( a ) and were stimulated with M-CSF for 1, 2 and 4 h. Total RNAs were isolated and subjected to Ets1 -, Ets2 -, Etv5 -, Egr1 -, Id1 -, Id3 -, HoxA1 - and GAPDH -specific reverse transcriptase (RT)-PCR. ( c ) Sister cultures of M-CSFR THOC5 (flox/flox) MEF were infected with Ad-GFP or Ad-GFP-Cre virus for 3 days. After serum starvation for 24 h, cells were stimulated with M-CSF for 1 and 2 h. Protein extract from cells before stimulation (0 h) were supplied for THOC5- and GAPDH-specific immunoblot (Blot). Total RNAs were isolated from each preparation and supplied for Ets1 -, Ets2 -, Egr1 -, GAPDH - and THOC5 -specific RT-PCR (RT-PCR). ( d ) Aliquots of cDNA samples from ( c ) were applied for quantitative RT-PCR analysis of Ets1 mRNA (TaqMan RT-PCR). Relative expression levels compared with GAPDH were normalized. Average values from four independent PCR reactions+S.D. are shown. ( e ) Cells were prepared as described in ( c ), but RNA and protein were isolated from the nuclear and the cytoplasmic fractions obtained from 5000 cells and then supplied for Ets1 -, Ets2 -, Egr1 -, and GAPDH -specific RT-PCR. Protein extracts were supplied for GAPDH- and Histone H3-specific immunoblot. ( f ) RNA was isolated from the nuclear fraction as described in ( a ) and subjected to Ets1 - and GAPDH -specific RT-PCR using an exon–intron primer pair as indicated. RNA samples were reverse transcribed using oligo dT or random hexamer (random) primer as indicated ( Supplementary Table S1 ). As negative controls, RNA samples were supplied for PCR without reverse transcription (−). We performed independent experiments, and we show one example of representative data ( a–f )

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Infection, Quantitative RT-PCR, Expressing, Polymerase Chain Reaction, Random Hexamer Labeling

6) Product Images from "PDGF-BB and TGF-?1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress"

Article Title: PDGF-BB and TGF-?1 on cross-talk between endothelial and smooth muscle cells in vascular remodeling induced by low shear stress

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

doi: 10.1073/pnas.1019219108

Incubation of TGF-β1 NAB in VSMCs increased the expression of lamin A in cocultured ECs, but PDGF-BB had no significant effect ( A ). NABs of both PDGF-BB and TGF-β1 incubation in VSMCs decreased expressions of LOX and phospho-ERK1/2 ( A
Figure Legend Snippet: Incubation of TGF-β1 NAB in VSMCs increased the expression of lamin A in cocultured ECs, but PDGF-BB had no significant effect ( A ). NABs of both PDGF-BB and TGF-β1 incubation in VSMCs decreased expressions of LOX and phospho-ERK1/2 ( A

Techniques Used: Incubation, Expressing

rPDGF-BB and rTGF-β1 decreased expression of lamin A and increased expression of LOX and phospho-ERK1/2 ( A ) and induced migration ( B ) and proliferation ( C ) of ECs. rPDGF-BB showed similar effects on VSMCs as in ECs ( D – F ). However, rTGF-β1
Figure Legend Snippet: rPDGF-BB and rTGF-β1 decreased expression of lamin A and increased expression of LOX and phospho-ERK1/2 ( A ) and induced migration ( B ) and proliferation ( C ) of ECs. rPDGF-BB showed similar effects on VSMCs as in ECs ( D – F ). However, rTGF-β1

Techniques Used: Expressing, Migration

In ECs and VSMCs, LowSS induced the secretion of PDGF-BB and TGF-β1 at 12 and 24 h ( A and B ) and decreased the expression of lamin A and LOX at 6, 12, and 24 h ( C and D ). The phosphorylation of ERK1/2 was up-regulated by LowSS at 6 and 12 h in
Figure Legend Snippet: In ECs and VSMCs, LowSS induced the secretion of PDGF-BB and TGF-β1 at 12 and 24 h ( A and B ) and decreased the expression of lamin A and LOX at 6, 12, and 24 h ( C and D ). The phosphorylation of ERK1/2 was up-regulated by LowSS at 6 and 12 h in

Techniques Used: Expressing

7) 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:

8) Product Images from "Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation"

Article Title: Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation

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

doi: 10.1152/ajplung.00048.2015

Levels of phosphorylated and total ERK1/2 protein within lungs subjected to 5 min of low- or high-stretch ventilation. Consistent with findings from Fig. 3 , levels of phospho-ERK1/2 clearly increased with high-stretch ventilation, whereas levels of total ERK1/2 within the same lungs were unaltered; n = 4. * P
Figure Legend Snippet: Levels of phosphorylated and total ERK1/2 protein within lungs subjected to 5 min of low- or high-stretch ventilation. Consistent with findings from Fig. 3 , levels of phospho-ERK1/2 clearly increased with high-stretch ventilation, whereas levels of total ERK1/2 within the same lungs were unaltered; n = 4. * P

Techniques Used:

9) Product Images from "Interleukin-32 Promotes Osteoclast Differentiation but Not Osteoclast Activation"

Article Title: Interleukin-32 Promotes Osteoclast Differentiation but Not Osteoclast Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004173

Effects of IL-32 on signalling pathways involved during osteoclastogenesis. Phosphorylation of ERK1/2, JNK, Akt and IkB-α in human PBMCs after 15 minutes exposure to RANKL or IL-32. β-actin was used an internal control of gel loading.
Figure Legend Snippet: Effects of IL-32 on signalling pathways involved during osteoclastogenesis. Phosphorylation of ERK1/2, JNK, Akt and IkB-α in human PBMCs after 15 minutes exposure to RANKL or IL-32. β-actin was used an internal control of gel loading.

Techniques Used:

Schematic representation of downstream pathways activated by RANKL or IL-32 treatment. The discrepancy observed between IL-32 and RANKL signalling pathways (i.e. increased ERK1/2 and Akt activation by IL-32) may lead to the activation of different downstream targets which in turn could contribute to the inability of cells to express F-actin ring and resorb in response to IL-32.
Figure Legend Snippet: Schematic representation of downstream pathways activated by RANKL or IL-32 treatment. The discrepancy observed between IL-32 and RANKL signalling pathways (i.e. increased ERK1/2 and Akt activation by IL-32) may lead to the activation of different downstream targets which in turn could contribute to the inability of cells to express F-actin ring and resorb in response to IL-32.

Techniques Used: Activation Assay

10) Product Images from "Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations"

Article Title: Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations

Journal: Nature genetics

doi: 10.1038/ng1586

Expression of mutant proteins in human melanocytes. ( A ) Experimental schematic. ( B ) Confirmation of protein expression; Ras=active N-Ras G12V , CDK4=active CDK4 R24C , p53 DN =dominant-negative p53 R248W . ( C ) Levels of active phosphorylated and total ERK1/2
Figure Legend Snippet: Expression of mutant proteins in human melanocytes. ( A ) Experimental schematic. ( B ) Confirmation of protein expression; Ras=active N-Ras G12V , CDK4=active CDK4 R24C , p53 DN =dominant-negative p53 R248W . ( C ) Levels of active phosphorylated and total ERK1/2

Techniques Used: Expressing, Mutagenesis, Dominant Negative Mutation

11) Product Images from "Impaired TrkB Signaling Underlies Reduced BDNF-Mediated Trophic Support of Striatal Neurons in the R6/2 Mouse Model of Huntington’s Disease"

Article Title: Impaired TrkB Signaling Underlies Reduced BDNF-Mediated Trophic Support of Striatal Neurons in the R6/2 Mouse Model of Huntington’s Disease

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2016.00037

TrkB activation and signal transduction is impaired in R6/2 striatal cultures. (A) Sample immunoblots of TrkB and Erk1/2 signaling proteins from WT and R6/2 cultures after exposure to control (−) or 100 ng/mL BDNF (+) supplemented media. (B) fl-TrkB and (C) Erk1/2 levels are similar in control treated WT ( n = 6) and R6/2 ( n = 5) cultures. BDNF treatment did not change the levels of these proteins. (D) p-TrkB and (E) p-Erk1/2 levels after treatment with saline or BDNF. BDNF treatment increased p-TrkB and p-Erk1/2 levels to a greater extent in WT ( n = 6) compared to R6/2 ( n = 5) cultures. Relative protein levels were determined by normalization to the neuronal specific cytoskeleton protein β-tubulin. Unpaired Student’s t -test * p
Figure Legend Snippet: TrkB activation and signal transduction is impaired in R6/2 striatal cultures. (A) Sample immunoblots of TrkB and Erk1/2 signaling proteins from WT and R6/2 cultures after exposure to control (−) or 100 ng/mL BDNF (+) supplemented media. (B) fl-TrkB and (C) Erk1/2 levels are similar in control treated WT ( n = 6) and R6/2 ( n = 5) cultures. BDNF treatment did not change the levels of these proteins. (D) p-TrkB and (E) p-Erk1/2 levels after treatment with saline or BDNF. BDNF treatment increased p-TrkB and p-Erk1/2 levels to a greater extent in WT ( n = 6) compared to R6/2 ( n = 5) cultures. Relative protein levels were determined by normalization to the neuronal specific cytoskeleton protein β-tubulin. Unpaired Student’s t -test * p

Techniques Used: Activation Assay, Transduction, Western Blot

12) Product Images from "Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway"

Article Title: Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

doi: 10.4196/kjpp.2016.20.4.399

Effects of imperatorin on PFHxS-induced ERK activation. Cells were treated with 300 µM PFHxS or DMSO as a vehicle control for 30 min in the presence or absence of (A) imperatorin (50, 100, 500 nM), (B) MK801 (1 µM), DTZ (10 µM), NFD (10 µM) or PD (50 µM). The levels of phosphorylatedand total protein of ERK1/2 were detected by Western blot analysis. The blots were reprobed with GAPDH. The blots represent three independent experiments. The densities of bands were measured and the fold increase in ratio pERK/ERK was presented as mean±SEM of three independent experiments. * p
Figure Legend Snippet: Effects of imperatorin on PFHxS-induced ERK activation. Cells were treated with 300 µM PFHxS or DMSO as a vehicle control for 30 min in the presence or absence of (A) imperatorin (50, 100, 500 nM), (B) MK801 (1 µM), DTZ (10 µM), NFD (10 µM) or PD (50 µM). The levels of phosphorylatedand total protein of ERK1/2 were detected by Western blot analysis. The blots were reprobed with GAPDH. The blots represent three independent experiments. The densities of bands were measured and the fold increase in ratio pERK/ERK was presented as mean±SEM of three independent experiments. * p

Techniques Used: Activation Assay, Western Blot

13) Product Images from "EphA2 Engages Git1 to Suppress Arf6 Activity Modulating Epithelial Cell-Cell Contacts"

Article Title: EphA2 Engages Git1 to Suppress Arf6 Activity Modulating Epithelial Cell-Cell Contacts

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E08-06-0549

Cell density- and calcium-dependent suppression of Arf6 and Erk1/2 activities in MDCK cells. (A) Arf6 and Erk1/2 activities in cells cultured under dense and sparse densities. Arf6 activities were measured by the GST-GGA pulldown method. Amounts of Arf6
Figure Legend Snippet: Cell density- and calcium-dependent suppression of Arf6 and Erk1/2 activities in MDCK cells. (A) Arf6 and Erk1/2 activities in cells cultured under dense and sparse densities. Arf6 activities were measured by the GST-GGA pulldown method. Amounts of Arf6

Techniques Used: Cell Culture

14) Product Images from "Signaling by FGF Receptor 2, Not FGF Receptor 1, Regulates Myelin Thickness through Activation of ERK1/2–MAPK, Which Promotes mTORC1 Activity in an Akt-Independent Manner"

Article Title: Signaling by FGF Receptor 2, Not FGF Receptor 1, Regulates Myelin Thickness through Activation of ERK1/2–MAPK, Which Promotes mTORC1 Activity in an Akt-Independent Manner

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3316-16.2017

Downregulation of p-mTOR, p-Raptor, and p-S6RP but not p-Akt in oligodendrocytes of mice lacking Fgfr2 and recovery upon ERK1/2 activation. A , Cervical spinal cord sections from P15 control, Fgfr2 cKO , Fgfr1/Fgfr2 dKO , Fgfr2-KO;Mek /+, and Fgfr1/Fgfr2-dKO;Mek
Figure Legend Snippet: Downregulation of p-mTOR, p-Raptor, and p-S6RP but not p-Akt in oligodendrocytes of mice lacking Fgfr2 and recovery upon ERK1/2 activation. A , Cervical spinal cord sections from P15 control, Fgfr2 cKO , Fgfr1/Fgfr2 dKO , Fgfr2-KO;Mek /+, and Fgfr1/Fgfr2-dKO;Mek

Techniques Used: Mouse Assay, Activation Assay

Reduction of myelin gene expression in mice lacking Fgfr2 is rescued by the elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , Immunoblotting of equal amounts of total proteins from homogenates of spinal cord white matter and quantification
Figure Legend Snippet: Reduction of myelin gene expression in mice lacking Fgfr2 is rescued by the elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , Immunoblotting of equal amounts of total proteins from homogenates of spinal cord white matter and quantification

Techniques Used: Expressing, Mouse Assay, Activity Assay

ERK1/2 activity is downregulated in oligodendrocytes in mice lacking Fgfr2 but not Fgfr1 . A , Transverse sections of cervical spinal cords at P15, immunolabeled for p-ERK1/2 or pan-ERK1/2, show strong oligodendrocyte-like cellular staining in the white
Figure Legend Snippet: ERK1/2 activity is downregulated in oligodendrocytes in mice lacking Fgfr2 but not Fgfr1 . A , Transverse sections of cervical spinal cords at P15, immunolabeled for p-ERK1/2 or pan-ERK1/2, show strong oligodendrocyte-like cellular staining in the white

Techniques Used: Activity Assay, Mouse Assay, Immunolabeling, Staining

Reduction of white matter size in mice lacking Fgfr2 is rescued by elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. Cervical spinal cord sections from 2-month-old control, Fgfr1 cKO , Fgfr2 cKO , and Fgfr1/2 dKO mice immunolabeled for MBP
Figure Legend Snippet: Reduction of white matter size in mice lacking Fgfr2 is rescued by elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. Cervical spinal cord sections from 2-month-old control, Fgfr1 cKO , Fgfr2 cKO , and Fgfr1/2 dKO mice immunolabeled for MBP

Techniques Used: Mouse Assay, Activity Assay, Immunolabeling

Myrf mRNA is selectively reduced in mice lacking Fgfr2 and is rescued by elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , B , qRT-PCR analysis of adult spinal cords at P15 shows significant downregulation of Myrf mRNA levels (blue bars)
Figure Legend Snippet: Myrf mRNA is selectively reduced in mice lacking Fgfr2 and is rescued by elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , B , qRT-PCR analysis of adult spinal cords at P15 shows significant downregulation of Myrf mRNA levels (blue bars)

Techniques Used: Mouse Assay, Activity Assay, Quantitative RT-PCR

Reduction of myelin sheath thickness in mice lacking Fgfr2 is rescued by the elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , EM images taken from similar ventral regions of cervical spinal cords at low and high magnification at 5
Figure Legend Snippet: Reduction of myelin sheath thickness in mice lacking Fgfr2 is rescued by the elevation of ERK1/2 activity in Fgfr2 -deficient oligodendrocytes. A , EM images taken from similar ventral regions of cervical spinal cords at low and high magnification at 5

Techniques Used: Mouse Assay, Activity Assay

Downregulation of p-mTOR, p-Raptor, p-p70S6K, and p-S6RP but not p-Akt in mice lacking ERK1/2. A , Cervical spinal cord sections from P30 control and ERK1/2 dKO mice analyzed by immunolabeling for p-Akt T308 , p-mTOR S2448 , p-Raptor S696 , p-S6RP S235/S236 ,
Figure Legend Snippet: Downregulation of p-mTOR, p-Raptor, p-p70S6K, and p-S6RP but not p-Akt in mice lacking ERK1/2. A , Cervical spinal cord sections from P30 control and ERK1/2 dKO mice analyzed by immunolabeling for p-Akt T308 , p-mTOR S2448 , p-Raptor S696 , p-S6RP S235/S236 ,

Techniques Used: Mouse Assay, Immunolabeling

15) Product Images from "Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS"

Article Title: Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0299-16.2016

Myelin/axonal pathology occur in the PNS by elevation of ERK1/2 activity in Schwann cells. A , Immunoblot analysis of phospho-ERK1/2 in lysates of sciatic nerves from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice at P 21 shows a statistically significant graded elevation of ERK1/2 activity with increased dosage of constitutively active Mek1 gene. Error bars represent SEM. ** p
Figure Legend Snippet: Myelin/axonal pathology occur in the PNS by elevation of ERK1/2 activity in Schwann cells. A , Immunoblot analysis of phospho-ERK1/2 in lysates of sciatic nerves from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice at P 21 shows a statistically significant graded elevation of ERK1/2 activity with increased dosage of constitutively active Mek1 gene. Error bars represent SEM. ** p

Techniques Used: Activity Assay, Mouse Assay

Increase in constitutively active Mek1 gene dosage in transgenic mice correlates with a graded increase in ERK1/2 activity and EGFP expression in oligodendrocytes. A , Transverse sections of spinal cords from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice show graded EGFP signal intensity in the white matter of spinal cords of transgenic but not control mice (top). Sections labeled for phospho-ERK1/2 (bottom) show increased cellular signal in the white matter of transgenic compared with control mice. B , Control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice were injected with tamoxifen either at 2 months or P10 for 8–10 d. Double immunolabeling for CC1 (red) and EGFP (green) at 1–2 MPI shows graded increase in EGFP expression in the spinal cords of PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice. C , Immunoblot analysis for phospho-ERK1/2 in lysates of spinal cord white matter from CnpCre;Mek /+, CnpCre;Mek/Mek , and littermate control mice or from PlpCre ERT ; Mek /+, PlpCre ERT ;Mek/Mek , and littermate control mice showing a statistically significant increase in its expression in the Mek/Mek compared with the Mek /+ or control mice. GAPDH is used as a loading control. Error bars indicate SEM. * p
Figure Legend Snippet: Increase in constitutively active Mek1 gene dosage in transgenic mice correlates with a graded increase in ERK1/2 activity and EGFP expression in oligodendrocytes. A , Transverse sections of spinal cords from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice show graded EGFP signal intensity in the white matter of spinal cords of transgenic but not control mice (top). Sections labeled for phospho-ERK1/2 (bottom) show increased cellular signal in the white matter of transgenic compared with control mice. B , Control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice were injected with tamoxifen either at 2 months or P10 for 8–10 d. Double immunolabeling for CC1 (red) and EGFP (green) at 1–2 MPI shows graded increase in EGFP expression in the spinal cords of PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice. C , Immunoblot analysis for phospho-ERK1/2 in lysates of spinal cord white matter from CnpCre;Mek /+, CnpCre;Mek/Mek , and littermate control mice or from PlpCre ERT ; Mek /+, PlpCre ERT ;Mek/Mek , and littermate control mice showing a statistically significant increase in its expression in the Mek/Mek compared with the Mek /+ or control mice. GAPDH is used as a loading control. Error bars indicate SEM. * p

Techniques Used: Transgenic Assay, Mouse Assay, Activity Assay, Expressing, Labeling, Injection, Immunolabeling

ERK1/2 overactivation upregulates phospho-mTOR in oligodendrocytes of the adult CNS. A , Spinal cord sections from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with tamoxifen at 2 months and analyzed at 1 MPI by immunolabeling for phospho-mTOR 2448 show that the intensity of cellular staining in the white matter increases in the PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek compared with control mice. Representative images from the analysis of three animals per genotype are shown. Arrowheads point to phospho-mTOR+ cell bodies B , Immunoblot analysis for phospho-mTOR 2448 in lysates of spinal cords white matter from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice show statistically significant increases in its expression in both the PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with controls and a trend toward an increase in the PlpCre ERT ; Mek/Mek compared with PlpCre ERT ; Mek /+ mice. GAPDH is shown as the loading control. Error bars indicate SEM. ** p
Figure Legend Snippet: ERK1/2 overactivation upregulates phospho-mTOR in oligodendrocytes of the adult CNS. A , Spinal cord sections from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with tamoxifen at 2 months and analyzed at 1 MPI by immunolabeling for phospho-mTOR 2448 show that the intensity of cellular staining in the white matter increases in the PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek compared with control mice. Representative images from the analysis of three animals per genotype are shown. Arrowheads point to phospho-mTOR+ cell bodies B , Immunoblot analysis for phospho-mTOR 2448 in lysates of spinal cords white matter from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice show statistically significant increases in its expression in both the PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with controls and a trend toward an increase in the PlpCre ERT ; Mek/Mek compared with PlpCre ERT ; Mek /+ mice. GAPDH is shown as the loading control. Error bars indicate SEM. ** p

Techniques Used: Mouse Assay, Injection, Immunolabeling, Staining, Expressing

Elevation of ERK1/2 activity during adulthood can reactivate quiescent mature oligodendrocytes to upregulate myelin gene expression and myelin growth. A , Control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with tamoxifen at 2 months of age, analyzed at 3 MPI by in situ hybridization, show that the signal intensity of MBP mRNA expression is increased in the spinal cords of PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with littermate controls. B , C , Quantification of MBP and PLP mRNA levels by qRT-PCR at 3, 6, and 8 MPI shows a significant increase in their levels in both PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with controls at all time points. Control values are normalized to 1. Error bars indicate SEM. * p
Figure Legend Snippet: Elevation of ERK1/2 activity during adulthood can reactivate quiescent mature oligodendrocytes to upregulate myelin gene expression and myelin growth. A , Control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with tamoxifen at 2 months of age, analyzed at 3 MPI by in situ hybridization, show that the signal intensity of MBP mRNA expression is increased in the spinal cords of PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with littermate controls. B , C , Quantification of MBP and PLP mRNA levels by qRT-PCR at 3, 6, and 8 MPI shows a significant increase in their levels in both PlpCre ERT ; Mek /+ and PlpCre ERT ; Mek/Mek mice compared with controls at all time points. Control values are normalized to 1. Error bars indicate SEM. * p

Techniques Used: Activity Assay, Expressing, Mouse Assay, Injection, In Situ Hybridization, Plasmid Purification, Quantitative RT-PCR

Incremental trend in myelin gene expression and size of oligodendrocytes upon graded upregulation of ERK1/2 activity. A , Transverse sections of cervical spinal cord from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice analyzed at 3 months (M) by in situ hybridization for MBP mRNA expression show elevated signal intensity in the CnpCre;Mek /+ compared with control mice, which appeared to be further increased in the CnpCre;Mek/Mek mice. B , Quantification of mRNA levels for MOG, MBP, MAG, and PLP by qRT-PCR show a statistically significant increase of MOG and a trend toward an increase of other transcripts in the CnpCre;Mek/Mek compared with CnpCre;Mek /+ mice. Compared with controls, both the transgenics show statistically significant increases. Error bars indicate SEM. * p
Figure Legend Snippet: Incremental trend in myelin gene expression and size of oligodendrocytes upon graded upregulation of ERK1/2 activity. A , Transverse sections of cervical spinal cord from control, CnpCre;Mek /+, and CnpCre;Mek/Mek mice analyzed at 3 months (M) by in situ hybridization for MBP mRNA expression show elevated signal intensity in the CnpCre;Mek /+ compared with control mice, which appeared to be further increased in the CnpCre;Mek/Mek mice. B , Quantification of mRNA levels for MOG, MBP, MAG, and PLP by qRT-PCR show a statistically significant increase of MOG and a trend toward an increase of other transcripts in the CnpCre;Mek/Mek compared with CnpCre;Mek /+ mice. Compared with controls, both the transgenics show statistically significant increases. Error bars indicate SEM. * p

Techniques Used: Expressing, Activity Assay, Mouse Assay, In Situ Hybridization, Plasmid Purification, Quantitative RT-PCR

Increasing the strength of ERK1/2 activation in oligodendrocytes results in myelin and axonal pathology at higher doses (Mek/Mek), but not at lower doses (Mek/+) regardless of the timing of its activation. A , Transverse semithin sections of ventral spinal cord from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with Tm at 2 months of age and analyzed at 3 and 8 MPI show abnormal myelin profiles with darkly stained ovals (red arrowheads) and degenerating axons, which often appeared as empty spaces surrounded by thin wraps of myelin (green asterisk) in the PlpCre ERT ; Mek/Mek , but not in the PlpCre ERT ; Mek /+ or control mice. High-magnification EM images of ventral spinal cords from PlpCre ERT ; Mek/Mek mice at 8 MPI show unmyelinated (red asterisk) and thinly myelinated axons ( Aa ), redundant or collapsed myelin profiles representing myelin remaining after axonal loss ( Ab ), and a degenerating axon with deteriorating myelin sheath ( Ac ). B , Quantification of darkly stained ovals in half sections of lateral–ventral (L–V) white matter of spinal cord from mice injected with Tm at 2 months and analyzed at 3 MPI show a statistically significant increase in the PlpCre ERT ; Mek/Mek mice compared with the PlpCre ERT ; Mek /+ and control mice. C , Quantification of myelinated and unmyelinated axons from EM images of ventral spinal cords shows that the percentage of unmyelinated axons is significantly higher in the PlpCre ERT ; Mek/Mek mice (∼31%) compared with control and PlpCre ERT ; Mek /+ mice (∼9%) and the number of myelinated axons is significantly lower; 911 (control), 1284 (Mek/+), and 1442 (Mek/Mek) axons from two animals of each group were examined. D , Transverse semithin sections of ventral spinal cord from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with Tm at P10 and analyzed at 8 MPI also show abnormal myelin profiles with darkly stained ovals (red arrowheads) and degenerating axons in the PlpCre ERT ; Mek/Mek , but not in the PlpCre ERT ; Mek /+ or control mice. High-magnification EM images from PlpCre ERT ; Mek/Mek ( Da – Dc ) show myelinated axonal profiles with axons in varying stages of degeneration. Multiple images of semithin and ultrathin sections from two to three mice per group were analyzed.
Figure Legend Snippet: Increasing the strength of ERK1/2 activation in oligodendrocytes results in myelin and axonal pathology at higher doses (Mek/Mek), but not at lower doses (Mek/+) regardless of the timing of its activation. A , Transverse semithin sections of ventral spinal cord from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with Tm at 2 months of age and analyzed at 3 and 8 MPI show abnormal myelin profiles with darkly stained ovals (red arrowheads) and degenerating axons, which often appeared as empty spaces surrounded by thin wraps of myelin (green asterisk) in the PlpCre ERT ; Mek/Mek , but not in the PlpCre ERT ; Mek /+ or control mice. High-magnification EM images of ventral spinal cords from PlpCre ERT ; Mek/Mek mice at 8 MPI show unmyelinated (red asterisk) and thinly myelinated axons ( Aa ), redundant or collapsed myelin profiles representing myelin remaining after axonal loss ( Ab ), and a degenerating axon with deteriorating myelin sheath ( Ac ). B , Quantification of darkly stained ovals in half sections of lateral–ventral (L–V) white matter of spinal cord from mice injected with Tm at 2 months and analyzed at 3 MPI show a statistically significant increase in the PlpCre ERT ; Mek/Mek mice compared with the PlpCre ERT ; Mek /+ and control mice. C , Quantification of myelinated and unmyelinated axons from EM images of ventral spinal cords shows that the percentage of unmyelinated axons is significantly higher in the PlpCre ERT ; Mek/Mek mice (∼31%) compared with control and PlpCre ERT ; Mek /+ mice (∼9%) and the number of myelinated axons is significantly lower; 911 (control), 1284 (Mek/+), and 1442 (Mek/Mek) axons from two animals of each group were examined. D , Transverse semithin sections of ventral spinal cord from control, PlpCre ERT ; Mek /+, and PlpCre ERT ; Mek/Mek mice injected with Tm at P10 and analyzed at 8 MPI also show abnormal myelin profiles with darkly stained ovals (red arrowheads) and degenerating axons in the PlpCre ERT ; Mek/Mek , but not in the PlpCre ERT ; Mek /+ or control mice. High-magnification EM images from PlpCre ERT ; Mek/Mek ( Da – Dc ) show myelinated axonal profiles with axons in varying stages of degeneration. Multiple images of semithin and ultrathin sections from two to three mice per group were analyzed.

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

Increased collagen deposition and mast cell infiltration occur in the sciatic nerves by superelevation of ERK1/2 activity in Schwann cells. A , Gross micrographs showing enlargement of sciatic nerves of CnpCre;Mek /+ and CnpCre;Mek/Mek mice compared with controls at 3 months of age (M). B , Collagen deposition, shown by Masson's trichrome staining (blue) in longitudinal sections of sciatic nerves at 3 months, is enhanced in the CnpCre;Mek/Mek compared with the CnpCre;Mek /+ mice. By 8 months, staining intensity also increases in the CnpCre;Mek /+ mice. EM image of sciatic nerve shows collagen fibers in the CnpCre;Mek/Mek mice. C , Mast cell infiltration, shown by Giemsa staining (blue) in longitudinal sections of sciatic nerves, is increased at 3 months in the CnpCre;Mek/Mek compared with the CnpCre;Mek /+ mice (number of mast cells/field: control, 0.6 ± 0.3; CnpCre;Mek /+, 5.5 ± 0.3; CnpCre;Mek/Mek , 9.9 ± 0.3. p
Figure Legend Snippet: Increased collagen deposition and mast cell infiltration occur in the sciatic nerves by superelevation of ERK1/2 activity in Schwann cells. A , Gross micrographs showing enlargement of sciatic nerves of CnpCre;Mek /+ and CnpCre;Mek/Mek mice compared with controls at 3 months of age (M). B , Collagen deposition, shown by Masson's trichrome staining (blue) in longitudinal sections of sciatic nerves at 3 months, is enhanced in the CnpCre;Mek/Mek compared with the CnpCre;Mek /+ mice. By 8 months, staining intensity also increases in the CnpCre;Mek /+ mice. EM image of sciatic nerve shows collagen fibers in the CnpCre;Mek/Mek mice. C , Mast cell infiltration, shown by Giemsa staining (blue) in longitudinal sections of sciatic nerves, is increased at 3 months in the CnpCre;Mek/Mek compared with the CnpCre;Mek /+ mice (number of mast cells/field: control, 0.6 ± 0.3; CnpCre;Mek /+, 5.5 ± 0.3; CnpCre;Mek/Mek , 9.9 ± 0.3. p

Techniques Used: Activity Assay, Mouse Assay, Staining

16) Product Images from "TGF? enforces activation of eukaryotic elongation factor-2 (eEF2) via inactivation of eEF2 kinase by p90 ribosomal S6 kinase (p90Rsk) to induce mesangial cell hypertrophy"

Article Title: TGF? enforces activation of eukaryotic elongation factor-2 (eEF2) via inactivation of eEF2 kinase by p90 ribosomal S6 kinase (p90Rsk) to induce mesangial cell hypertrophy

Journal: FEBS letters

doi: 10.1016/j.febslet.2010.09.010

TGFβ stimulates p90Rsk in Erk1/2-dependent manner. (A) Lysates of mesangial cells incubated with 2 ng/ml TGFβ for the indicated periods of time were immunoblotted with the indicated antibodies. (B) Mesangial cells were treated with U0126
Figure Legend Snippet: TGFβ stimulates p90Rsk in Erk1/2-dependent manner. (A) Lysates of mesangial cells incubated with 2 ng/ml TGFβ for the indicated periods of time were immunoblotted with the indicated antibodies. (B) Mesangial cells were treated with U0126

Techniques Used: Incubation

TGFβ decreases phosphorylation of eEF2 by increasing phosphorylation of eEF2K in Erk1/2-dependent manner. (A and B) Lysates of mesangial cells incubated with 2 ng/ml TGFβ for the indicated periods of time were immunoblotted with the indicated
Figure Legend Snippet: TGFβ decreases phosphorylation of eEF2 by increasing phosphorylation of eEF2K in Erk1/2-dependent manner. (A and B) Lysates of mesangial cells incubated with 2 ng/ml TGFβ for the indicated periods of time were immunoblotted with the indicated

Techniques Used: Incubation

17) Product Images from "Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages *"

Article Title: Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.461004

Comparison of phosphorylation status of p38 MAPK and ERK1/2 mediated by Mtbhsp60 between the TLR2 receptor that undergoes endocytosis against TLR2 or TLR4 that does not undergo endocytosis. A, PMA-differentiated THP-1 macrophages were pretreated with
Figure Legend Snippet: Comparison of phosphorylation status of p38 MAPK and ERK1/2 mediated by Mtbhsp60 between the TLR2 receptor that undergoes endocytosis against TLR2 or TLR4 that does not undergo endocytosis. A, PMA-differentiated THP-1 macrophages were pretreated with

Techniques Used:

18) Product Images from "Carbonic Anhydrase Activation Is Associated With Worsened Pathological Remodeling in Human Ischemic Diabetic Cardiomyopathy"

Article Title: Carbonic Anhydrase Activation Is Associated With Worsened Pathological Remodeling in Human Ischemic Diabetic Cardiomyopathy

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.113.000434

MicroR‐23b (miR‐23b) downregulation is dependent on p38 MAPK pathway. A, p38 MAPK, ERK1/2, and JNK were significantly more phosphorylated in the myocardial samples from T2‐DM compared with NDM as shown by representative Western blots and O.D. semiquantitative analysis; * P
Figure Legend Snippet: MicroR‐23b (miR‐23b) downregulation is dependent on p38 MAPK pathway. A, p38 MAPK, ERK1/2, and JNK were significantly more phosphorylated in the myocardial samples from T2‐DM compared with NDM as shown by representative Western blots and O.D. semiquantitative analysis; * P

Techniques Used: Western Blot

19) Product Images from "PKC? regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)"

Article Title: PKC? regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200108062

Overexpression of PKCα and ɛ induces ERK1/2 phosphorylation. (A) Western blot analysis for ERK1/2, p38, and JNK wild-type and phosphorylated forms in AdPKC-infected cardiomyocytes. Anisomycin was used as a control for JNK and p38 activation only. (B) Quantitation from three independent experiments demonstrates significant ERK1/2 activation induced by AdPKCα and ɛ infection compared with Adβgal infection. * P
Figure Legend Snippet: Overexpression of PKCα and ɛ induces ERK1/2 phosphorylation. (A) Western blot analysis for ERK1/2, p38, and JNK wild-type and phosphorylated forms in AdPKC-infected cardiomyocytes. Anisomycin was used as a control for JNK and p38 activation only. (B) Quantitation from three independent experiments demonstrates significant ERK1/2 activation induced by AdPKCα and ɛ infection compared with Adβgal infection. * P

Techniques Used: Over Expression, Western Blot, Infection, Activation Assay, Quantitation Assay

AdPKCαdn inhibits PMA-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) Western blot analysis of phosphorylated ERK1/2 from AdPKCαdn-, AdPKCβIIdn-, AdPKCδdn-, and AdPKCɛdn-infected cardiomyocytes before and after PMA treatment. The amount of total ERK1/2 did not vary. (B) Histogram showing a significant decrease in phosphorylated ERK1/2 only with AdPKCαdn infection ( n = 3). * P
Figure Legend Snippet: AdPKCαdn inhibits PMA-induced ERK1/2 phosphorylation in neonatal cardiomyocytes. (A) Western blot analysis of phosphorylated ERK1/2 from AdPKCαdn-, AdPKCβIIdn-, AdPKCδdn-, and AdPKCɛdn-infected cardiomyocytes before and after PMA treatment. The amount of total ERK1/2 did not vary. (B) Histogram showing a significant decrease in phosphorylated ERK1/2 only with AdPKCαdn infection ( n = 3). * P

Techniques Used: Western Blot, Infection

20) Product Images from "Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis"

Article Title: Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00559.2013

VEGF-C stimulates ERK phosphorylation in myofibroblasts. Compared with controls, VEGF-C elevated ERK phosphorylation (p-ERK1/2), which was blocked by the ERK inhibitor U0126 ( A ). VEGF-C-induced myofibroblast proliferation ( B ) and type I collagen synthesis
Figure Legend Snippet: VEGF-C stimulates ERK phosphorylation in myofibroblasts. Compared with controls, VEGF-C elevated ERK phosphorylation (p-ERK1/2), which was blocked by the ERK inhibitor U0126 ( A ). VEGF-C-induced myofibroblast proliferation ( B ) and type I collagen synthesis

Techniques Used:

21) Product Images from "Rhamm−/− fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair"

Article Title: Rhamm−/− fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200511027

ERK1,2 activation is aberrant in Rhamm −/− wound granulation tissue. Both Wt and Rh −/− granulation tissue fibroblasts are positive for phospho-ERK1,2 on day 3 after wounding (brown). ERK1,2 activity significantly increases in Wt wound granulation tissue by day 7 and drops to background by day 14. ERK1,2 activity prematurely drops in Rh −/− wound granulation tissue to background at day 7 and remains low at day 14. Mean and SEM; n = 15 images of three serial sections of wounds from five Wt and Rh −/− mice. *, P
Figure Legend Snippet: ERK1,2 activation is aberrant in Rhamm −/− wound granulation tissue. Both Wt and Rh −/− granulation tissue fibroblasts are positive for phospho-ERK1,2 on day 3 after wounding (brown). ERK1,2 activity significantly increases in Wt wound granulation tissue by day 7 and drops to background by day 14. ERK1,2 activity prematurely drops in Rh −/− wound granulation tissue to background at day 7 and remains low at day 14. Mean and SEM; n = 15 images of three serial sections of wounds from five Wt and Rh −/− mice. *, P

Techniques Used: Activation Assay, Activity Assay, Mouse Assay

Serum-induced ERK1,2 activation is defective in Rhamm −/− fibroblasts. (A) ELISA of total cellular phospho-ERK1,2. Active ERK1,2 levels are significantly higher after serum stimulation in Rh FL -rescued than in Rh −/− fibroblasts. Values at 0 min were subtracted from values at 30 and 60 min. Mean and SEM; n = 3 replicates. One of three experiments is shown. (B) Western blots of phospho-ERK1,2. Rh FL -rescued fibroblasts sustain serum-induced ERK1,2 activity for 50 min compared with 10 min in Rh −/− fibroblasts. One of five experiments is shown. (C) Confocal micrographs and image analysis of phospho-ERK1,2. Overall levels and nuclear (blue) targeting of phospho-ERK1,2 (red) are reduced in Rh −/− compared with Rh FL -rescued fibroblasts. Mean and SEM; n = 15 cells for each time point. One of four experiments is shown. *, P
Figure Legend Snippet: Serum-induced ERK1,2 activation is defective in Rhamm −/− fibroblasts. (A) ELISA of total cellular phospho-ERK1,2. Active ERK1,2 levels are significantly higher after serum stimulation in Rh FL -rescued than in Rh −/− fibroblasts. Values at 0 min were subtracted from values at 30 and 60 min. Mean and SEM; n = 3 replicates. One of three experiments is shown. (B) Western blots of phospho-ERK1,2. Rh FL -rescued fibroblasts sustain serum-induced ERK1,2 activity for 50 min compared with 10 min in Rh −/− fibroblasts. One of five experiments is shown. (C) Confocal micrographs and image analysis of phospho-ERK1,2. Overall levels and nuclear (blue) targeting of phospho-ERK1,2 (red) are reduced in Rh −/− compared with Rh FL -rescued fibroblasts. Mean and SEM; n = 15 cells for each time point. One of four experiments is shown. *, P

Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Cell surface Rhamm rescues motility of Rhamm −/− fibroblasts. (A) Motility in response to Rhamm beads. Rh −/− fibroblast motility is significantly increased when cells contact recombinant Rhamm beads, compared with control GST beads. Rhamm bead–stimulated motility is similar to Rh FL -rescued fibroblast motility and is blocked by a Mek1 inhibitor and CD44 antibody. Mean and SEM; n = 30 cells. One of eight experiments is shown. (B) ERK1,2 activation in response to Rhamm beads. Serum-induced ERK1,2 activity (red; white in insets) in Rh −/− fibroblasts is significantly stimulated by Rhamm beads but not control GST beads. ERK1,2 activity is not increased in Rh −/− fibroblasts when CD44 (green) is not expressed (Rh −/− :CD44 −/− ). Rhamm beads do not increase nuclear phospho-ERK1,2 in Rh −/− :CD44 −/− fibroblasts. Mean and SEM; n = 25 cells. One of three experiments is shown. *, P
Figure Legend Snippet: Cell surface Rhamm rescues motility of Rhamm −/− fibroblasts. (A) Motility in response to Rhamm beads. Rh −/− fibroblast motility is significantly increased when cells contact recombinant Rhamm beads, compared with control GST beads. Rhamm bead–stimulated motility is similar to Rh FL -rescued fibroblast motility and is blocked by a Mek1 inhibitor and CD44 antibody. Mean and SEM; n = 30 cells. One of eight experiments is shown. (B) ERK1,2 activation in response to Rhamm beads. Serum-induced ERK1,2 activity (red; white in insets) in Rh −/− fibroblasts is significantly stimulated by Rhamm beads but not control GST beads. ERK1,2 activity is not increased in Rh −/− fibroblasts when CD44 (green) is not expressed (Rh −/− :CD44 −/− ). Rhamm beads do not increase nuclear phospho-ERK1,2 in Rh −/− :CD44 −/− fibroblasts. Mean and SEM; n = 25 cells. One of three experiments is shown. *, P

Techniques Used: Recombinant, Activation Assay, Activity Assay

Cell surface Rhamm is required for CD44 display and complexing with phospho-ERK1,2. (A) Effect of Rhamm and ERK1,2 activity on surface display of CD44. (a and d) Rh −/− fibroblasts; (b–f) Rh FL -rescued fibroblasts. Live-cell immunofluorescence shows that Rh −/− fibroblasts exhibit less surface CD44 than Rh FL -rescued fibroblasts. A Mek1 inhibitor and Rhamm antibody block CD44 cell surface display. Recombinant Rhamm beads rescue display on Rh −/− cells in contact with or close to beads. One of three experiments is shown. (B) Effect of Rhamm on CD44 and phospho-ERK1,2 colocalization. Confocal micrographs and image analysis show that serum stimulates greater colocalization (white) of CD44 (green) and phospho-ERK1,2 (red) in Rh FL -rescued than Rh −/− fibroblasts, and colocalization is reduced by Rhamm antibody. Mean and SEM; n = 25 cells. One of four experiments is shown. *, P
Figure Legend Snippet: Cell surface Rhamm is required for CD44 display and complexing with phospho-ERK1,2. (A) Effect of Rhamm and ERK1,2 activity on surface display of CD44. (a and d) Rh −/− fibroblasts; (b–f) Rh FL -rescued fibroblasts. Live-cell immunofluorescence shows that Rh −/− fibroblasts exhibit less surface CD44 than Rh FL -rescued fibroblasts. A Mek1 inhibitor and Rhamm antibody block CD44 cell surface display. Recombinant Rhamm beads rescue display on Rh −/− cells in contact with or close to beads. One of three experiments is shown. (B) Effect of Rhamm on CD44 and phospho-ERK1,2 colocalization. Confocal micrographs and image analysis show that serum stimulates greater colocalization (white) of CD44 (green) and phospho-ERK1,2 (red) in Rh FL -rescued than Rh −/− fibroblasts, and colocalization is reduced by Rhamm antibody. Mean and SEM; n = 25 cells. One of four experiments is shown. *, P

Techniques Used: Activity Assay, Immunofluorescence, Blocking Assay, Recombinant

CD44 coassociates with Rhamm and active ERK1,2. (A) CD44 protein expression and distribution. Rh −/− and Wt fibroblasts express similar levels of CD44 proteins (β-actin loading control). One of three experiments is shown. Confocal analysis shows that CD44 (green) occurs mainly in vesicles with phospho-ERK1,2 (red; colocalization shown as white) after Rh FL rescue. CD44 staining is amorphous and not associated with phospho-ERK1,2 in Rh −/− fibroblasts. One of four experiments is shown. (B) Rhamm, CD44, and ERK1,2 form complexes. Recombinant Rhamm-GST protein–Sepharose (Rhamm-GST beads) pulls down CD44s, a possible CD44 variant (120 kD), and ERK1,2. One of three experiments is shown. Confocal analysis confirms that Rhamm (red) and CD44 (green) colocalize (white) in processes of Rh FL -rescued fibroblasts (no Triton X-100) and in vesicles (+Triton X-100). One of three experiments is shown.
Figure Legend Snippet: CD44 coassociates with Rhamm and active ERK1,2. (A) CD44 protein expression and distribution. Rh −/− and Wt fibroblasts express similar levels of CD44 proteins (β-actin loading control). One of three experiments is shown. Confocal analysis shows that CD44 (green) occurs mainly in vesicles with phospho-ERK1,2 (red; colocalization shown as white) after Rh FL rescue. CD44 staining is amorphous and not associated with phospho-ERK1,2 in Rh −/− fibroblasts. One of four experiments is shown. (B) Rhamm, CD44, and ERK1,2 form complexes. Recombinant Rhamm-GST protein–Sepharose (Rhamm-GST beads) pulls down CD44s, a possible CD44 variant (120 kD), and ERK1,2. One of three experiments is shown. Confocal analysis confirms that Rhamm (red) and CD44 (green) colocalize (white) in processes of Rh FL -rescued fibroblasts (no Triton X-100) and in vesicles (+Triton X-100). One of three experiments is shown.

Techniques Used: Expressing, Staining, Recombinant, Variant Assay

Cell surface Rhamm (CD168) and CD44 are required for ERK1,2 activity and motility in response to FCS and HA. (A) Role of cell surface Rhamm in nuclear ERK1,2 activity. Rhamm antibody significantly reduces levels of serum-induced nuclear phospho-ERK1,2. Mean and SEM; n = 25 cells. One of four experiments is shown. (B) Role of CD44 in nuclear ERK1,2 activity. CD44 antibody significantly reduces the levels of serum-induced nuclear phospho-ERK1,2. Mean and SEM; n = 25 cells. One of four experiments is shown. (C) Motility in response to FCS. Rh FL expression in Rh −/− fibroblasts significantly increases motility. The dotted line is Rh −/− fibroblast motility, which did not vary with treatment. Rh FL rescue requires surface CD44 and ERK1,2 activity, as CD44 antibody or a Mek1 inhibitor (UO126) significantly reduces motility. Rhamm antibody blocks Rh FL -rescued but not Rh −/− fibroblast motility. Mean and SEM; n = 30 cells. One of six experiments is shown. (D) Motility in response to HA. Wt, but not Rh −/− , fibroblasts increase random motility in response to HA. Rhamm antibody reduces HA-mediated motility of Wt but not Rh −/− fibroblasts. Fibroblasts were first exposed to PMA to generate responsiveness to HA. Mean and SEM; n = 30 cells. One of four experiments is shown. *, P
Figure Legend Snippet: Cell surface Rhamm (CD168) and CD44 are required for ERK1,2 activity and motility in response to FCS and HA. (A) Role of cell surface Rhamm in nuclear ERK1,2 activity. Rhamm antibody significantly reduces levels of serum-induced nuclear phospho-ERK1,2. Mean and SEM; n = 25 cells. One of four experiments is shown. (B) Role of CD44 in nuclear ERK1,2 activity. CD44 antibody significantly reduces the levels of serum-induced nuclear phospho-ERK1,2. Mean and SEM; n = 25 cells. One of four experiments is shown. (C) Motility in response to FCS. Rh FL expression in Rh −/− fibroblasts significantly increases motility. The dotted line is Rh −/− fibroblast motility, which did not vary with treatment. Rh FL rescue requires surface CD44 and ERK1,2 activity, as CD44 antibody or a Mek1 inhibitor (UO126) significantly reduces motility. Rhamm antibody blocks Rh FL -rescued but not Rh −/− fibroblast motility. Mean and SEM; n = 30 cells. One of six experiments is shown. (D) Motility in response to HA. Wt, but not Rh −/− , fibroblasts increase random motility in response to HA. Rhamm antibody reduces HA-mediated motility of Wt but not Rh −/− fibroblasts. Fibroblasts were first exposed to PMA to generate responsiveness to HA. Mean and SEM; n = 30 cells. One of four experiments is shown. *, P

Techniques Used: Activity Assay, Expressing

Mutant active Mek1 rescues ERK1,2 activity, motility, and CD44 surface display in Rh −/− fibroblasts. (A) ELISA of active ERK1,2. Expression of Mek1 in Rh −/− fibroblasts restores serum-induced ERK1,2 activity. Values at 0 min were subtracted from values at 30 and 60 min. Mean and SEM; n = 3 replicates from one of three experiments. (B) Western blot of active ERK1,2. Western blots confirm rescue of ERK1,2 activity by Mek1 in Rh −/− fibroblasts. One of five experiments is shown. (C) Motility and cell surface CD44 display. Expression of Mek1 in Rh −/− fibroblasts significantly increases motility, which is blocked by anti-CD44 antibody. Mean and SEM; n = 30 cells from one of three experiments. Live-cell immunofluorescence shows that Mek1 expression restores surface CD44 display in Rh −/− fibroblasts. The specificity of the anti-CD44 antibody is demonstrated by a lack of fluorescence in murine CD44 −/− :Rh −/− fibroblasts. One of three experiments is shown. *, P
Figure Legend Snippet: Mutant active Mek1 rescues ERK1,2 activity, motility, and CD44 surface display in Rh −/− fibroblasts. (A) ELISA of active ERK1,2. Expression of Mek1 in Rh −/− fibroblasts restores serum-induced ERK1,2 activity. Values at 0 min were subtracted from values at 30 and 60 min. Mean and SEM; n = 3 replicates from one of three experiments. (B) Western blot of active ERK1,2. Western blots confirm rescue of ERK1,2 activity by Mek1 in Rh −/− fibroblasts. One of five experiments is shown. (C) Motility and cell surface CD44 display. Expression of Mek1 in Rh −/− fibroblasts significantly increases motility, which is blocked by anti-CD44 antibody. Mean and SEM; n = 30 cells from one of three experiments. Live-cell immunofluorescence shows that Mek1 expression restores surface CD44 display in Rh −/− fibroblasts. The specificity of the anti-CD44 antibody is demonstrated by a lack of fluorescence in murine CD44 −/− :Rh −/− fibroblasts. One of three experiments is shown. *, P

Techniques Used: Mutagenesis, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunofluorescence, Fluorescence

22) Product Images from "Neurexin-Neuroligin Synaptic Complex Regulates Schizophrenia-Related DISC1/Kal-7/Rac1 “Signalosome”"

Article Title: Neurexin-Neuroligin Synaptic Complex Regulates Schizophrenia-Related DISC1/Kal-7/Rac1 “Signalosome”

Journal: Neural Plasticity

doi: 10.1155/2015/167308

NX1 β activates ERK1/2 and Akt kinases. Cortical neurons were grown for 8 days, subsequently treated with different concentrations of recombinant NX1 β for 5 min, and further immunoblotted for the detection of (a) ERK1/2, (b) p38 MAPK, and (c) Akt. (d) Effect of the PKA inhibitor H89 on NX1 β -induced ERK1/2 phosphorylation. The results from n ≥ 4 experiments are expressed as a percentage ± SEM. ∗ P
Figure Legend Snippet: NX1 β activates ERK1/2 and Akt kinases. Cortical neurons were grown for 8 days, subsequently treated with different concentrations of recombinant NX1 β for 5 min, and further immunoblotted for the detection of (a) ERK1/2, (b) p38 MAPK, and (c) Akt. (d) Effect of the PKA inhibitor H89 on NX1 β -induced ERK1/2 phosphorylation. The results from n ≥ 4 experiments are expressed as a percentage ± SEM. ∗ P

Techniques Used: Recombinant

23) Product Images from "Expression of ANO1/DOG1 is associated with shorter survival and progression of breast carcinomas"

Article Title: Expression of ANO1/DOG1 is associated with shorter survival and progression of breast carcinomas

Journal: Oncotarget

doi: 10.18632/oncotarget.23078

The expression of ANO1 is associated with the expression of signaling molecules associated with the proliferation and invasiveness of breast cancer cells ( A ) The protein levels of β-catenin, cyclin D1, MMP9, snail, N-cadherin, phospho-p38 MAPK, phospho-ERK1/2, NFκB p50, NFκB p65, and MYC decreased with knock-down of ANO1 by siRNA for ANO1 in both MCF7 and MDA-MB-231 cells. In contrast, the expression of the protein of E-cadherin increased with knock-down of ANO1 in both MCF7 and MDA-MB-231 cells. ( B ) The mRNA levels of β-catenin, cyclin D1, MMP9, snail, N-cadherin, NFκB p50, NFκB p65, and MYC decreased, but the expression of E-cadherin mRNA increased with knock-down of ANO1 in both MCF7 and MDA-MB-231 cells. ** P
Figure Legend Snippet: The expression of ANO1 is associated with the expression of signaling molecules associated with the proliferation and invasiveness of breast cancer cells ( A ) The protein levels of β-catenin, cyclin D1, MMP9, snail, N-cadherin, phospho-p38 MAPK, phospho-ERK1/2, NFκB p50, NFκB p65, and MYC decreased with knock-down of ANO1 by siRNA for ANO1 in both MCF7 and MDA-MB-231 cells. In contrast, the expression of the protein of E-cadherin increased with knock-down of ANO1 in both MCF7 and MDA-MB-231 cells. ( B ) The mRNA levels of β-catenin, cyclin D1, MMP9, snail, N-cadherin, NFκB p50, NFκB p65, and MYC decreased, but the expression of E-cadherin mRNA increased with knock-down of ANO1 in both MCF7 and MDA-MB-231 cells. ** P

Techniques Used: Expressing, Multiple Displacement Amplification

24) Product Images from "Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages"

Article Title: Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Journal: Pharmaceutical Biology

doi: 10.1080/13880209.2017.1357737

Effects of PFE on phosphorylation of MAPKs activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to LPS for 30 min. Total cellular proteins of cells were harvested for measurements of total or phosphorylated ERK1/2, JNK, and p38 by Western blotting. Data show mean ± SEM values of three independent experiments. * p
Figure Legend Snippet: Effects of PFE on phosphorylation of MAPKs activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to LPS for 30 min. Total cellular proteins of cells were harvested for measurements of total or phosphorylated ERK1/2, JNK, and p38 by Western blotting. Data show mean ± SEM values of three independent experiments. * p

Techniques Used: Activity Assay, Western Blot

25) Product Images from "A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease"

Article Title: A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease

Journal: The EMBO Journal

doi: 10.15252/embj.2018100101

CFTR malfunction drives P31–43‐induced epithelial stress response Immunoblot of PPARγ or phospho‐ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β‐actin. Mean ± SD of triplicates of independent experiments. ** P
Figure Legend Snippet: CFTR malfunction drives P31–43‐induced epithelial stress response Immunoblot of PPARγ or phospho‐ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β‐actin. Mean ± SD of triplicates of independent experiments. ** P

Techniques Used:

26) Product Images from "Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma"

Article Title: Synergistic antitumor effect of a γ-secretase inhibitor PF-03084014 and sorafenib in hepatocellular carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.26209

Enhanced Notch1 and Snail1 expression and EMT-mediated stemness in sorafenib resistant HCC spheroids ( A ) 97H spheroids were treated with high doses of sorafenib (10–15 μM) for over two weeks to generate sorafenib resistant cells. Western blot analysis of phospho-Erk1/2 and phospho-Akt in sorafenib-resistant (Sor+) cells compared with control (Sor−). ( B ) Western blot analysis of Snail1 and pStat3 in sorafenib-resistant (Sor+) cells compared with control (Sor−) (left panel). mRNA levels of NOTCH1 and its ligands JAG1 in sorafenib-resistant spheroids versus control (right panel). ( C ) mRNA levels of the EMT related genes SNAIL1, SNAIL2, CDH2 (N-CADHERIN), VIM (VIMENTIN) (left panel), and CDH1 (E-CADHERIN) (right panel) in sorafenib-resistant 97H spheroids compared to control (non-sorafenib resistance). ( D ) Phase contrast images of the cell morphologies of sorafenib-resistant cells compared with control. ( E ) mRNA levels of the multidrug resistant genes, ABCG2 and ABCB1, in sorafenib-resistant 97H spheroids versus control. ( F ) mRNA levels of the stemness genes, NANOG, OCT4, SOX2, and KLF4, in sorafenib-resistant and control spheroids. qPCR data are represented as the mean ± SD, n = 2 (from different sorafenib-resistant populations). An independent t test was used for statistical comparison. * p
Figure Legend Snippet: Enhanced Notch1 and Snail1 expression and EMT-mediated stemness in sorafenib resistant HCC spheroids ( A ) 97H spheroids were treated with high doses of sorafenib (10–15 μM) for over two weeks to generate sorafenib resistant cells. Western blot analysis of phospho-Erk1/2 and phospho-Akt in sorafenib-resistant (Sor+) cells compared with control (Sor−). ( B ) Western blot analysis of Snail1 and pStat3 in sorafenib-resistant (Sor+) cells compared with control (Sor−) (left panel). mRNA levels of NOTCH1 and its ligands JAG1 in sorafenib-resistant spheroids versus control (right panel). ( C ) mRNA levels of the EMT related genes SNAIL1, SNAIL2, CDH2 (N-CADHERIN), VIM (VIMENTIN) (left panel), and CDH1 (E-CADHERIN) (right panel) in sorafenib-resistant 97H spheroids compared to control (non-sorafenib resistance). ( D ) Phase contrast images of the cell morphologies of sorafenib-resistant cells compared with control. ( E ) mRNA levels of the multidrug resistant genes, ABCG2 and ABCB1, in sorafenib-resistant 97H spheroids versus control. ( F ) mRNA levels of the stemness genes, NANOG, OCT4, SOX2, and KLF4, in sorafenib-resistant and control spheroids. qPCR data are represented as the mean ± SD, n = 2 (from different sorafenib-resistant populations). An independent t test was used for statistical comparison. * p

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

27) Product Images from "Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway"

Article Title: Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00541

Role of Raf, Mek, and Erk isoforms in the MAPK pathway in H. pylori -induced gastrin promoter activation. ( A–E , upper panels) G240-Luc cells were transfected with 100 pM of NT siRNA, (A) A-Raf, B-Raf, or C-Raf siRNA, (B) Mek1, Mek2, or both Mek1 and Mek2 (Mek1/2) siRNA, and (C–E) Erk1, Erk2, or both Erk1 and Erk2 (Erk1/2) siRNA. Forty-eight hours post transfection, cells were infected with (A–C) H. pylori strain G27, (D) PMSS1, or (E) G27Δ cagA for 5 h. Luciferase activity is presented as a relative ratio to that of NT siRNA-transfected cells. The mean values ± SD of three separate experiments performed in triplicate are shown. * P
Figure Legend Snippet: Role of Raf, Mek, and Erk isoforms in the MAPK pathway in H. pylori -induced gastrin promoter activation. ( A–E , upper panels) G240-Luc cells were transfected with 100 pM of NT siRNA, (A) A-Raf, B-Raf, or C-Raf siRNA, (B) Mek1, Mek2, or both Mek1 and Mek2 (Mek1/2) siRNA, and (C–E) Erk1, Erk2, or both Erk1 and Erk2 (Erk1/2) siRNA. Forty-eight hours post transfection, cells were infected with (A–C) H. pylori strain G27, (D) PMSS1, or (E) G27Δ cagA for 5 h. Luciferase activity is presented as a relative ratio to that of NT siRNA-transfected cells. The mean values ± SD of three separate experiments performed in triplicate are shown. * P

Techniques Used: Activation Assay, Transfection, Infection, Luciferase, Activity Assay

28) Product Images from "Delphinidin Inhibits LPS-Induced MUC8 and MUC5B Expression Through Toll-like Receptor 4-Mediated ERK1/2 and p38 MAPK in Human Airway Epithelial Cells"

Article Title: Delphinidin Inhibits LPS-Induced MUC8 and MUC5B Expression Through Toll-like Receptor 4-Mediated ERK1/2 and p38 MAPK in Human Airway Epithelial Cells

Journal: Clinical and Experimental Otorhinolaryngology

doi: 10.3342/ceo.2014.7.3.198

The effects of delphinidin on LPS-induced phosphorylation of ERK1/2 and p38 MAPK in NCI-H292 cells. (A) Results of Western blot showed that LPS significantly activated the phosphorylation of ERK1/2 and p38 MAPK. (B) Results of Western blot showed that LPS-induced phosphorylation of ERK1/2 and p38 MAPK were significantly blocked by pretreatment with delphinidin for 60 minutes before being incubated with LPS for 60 minutes. ERK1/2, extracellular signal related kinase 1/2; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide. * P
Figure Legend Snippet: The effects of delphinidin on LPS-induced phosphorylation of ERK1/2 and p38 MAPK in NCI-H292 cells. (A) Results of Western blot showed that LPS significantly activated the phosphorylation of ERK1/2 and p38 MAPK. (B) Results of Western blot showed that LPS-induced phosphorylation of ERK1/2 and p38 MAPK were significantly blocked by pretreatment with delphinidin for 60 minutes before being incubated with LPS for 60 minutes. ERK1/2, extracellular signal related kinase 1/2; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide. * P

Techniques Used: Western Blot, Incubation

29) Product Images from "miR-21 and KLF4 jointly augment epithelial-mesenchymal transition via the Akt/ERK1/2 pathway"

Article Title: miR-21 and KLF4 jointly augment epithelial-mesenchymal transition via the Akt/ERK1/2 pathway

Journal: International Journal of Oncology

doi: 10.3892/ijo.2017.3876

Effects of miR-21 on the expression of KLF4, E-cadherin, N-cadherin, vimentin and/or Akt/ERK1/2 pathways. (A) The relative levels of p-Akt, Akt, p-ERK, ERK, KLF4 and EMT marker proteins were measured by western blot analysis. (B-D) Bands were semi-quantified using Quantity One software. (E) Western blot analysis showing protein levels of p-Akt, Akt, p-ERK1/2, ERK1/2, KLF4, mesenchymal markers (N-cadherin and vimentin) and epithelial cell marker (E-cadherin). (F-I) Bands were semi-quantified using Quantity One software. GAPDH was used as loading control ( ** P
Figure Legend Snippet: Effects of miR-21 on the expression of KLF4, E-cadherin, N-cadherin, vimentin and/or Akt/ERK1/2 pathways. (A) The relative levels of p-Akt, Akt, p-ERK, ERK, KLF4 and EMT marker proteins were measured by western blot analysis. (B-D) Bands were semi-quantified using Quantity One software. (E) Western blot analysis showing protein levels of p-Akt, Akt, p-ERK1/2, ERK1/2, KLF4, mesenchymal markers (N-cadherin and vimentin) and epithelial cell marker (E-cadherin). (F-I) Bands were semi-quantified using Quantity One software. GAPDH was used as loading control ( ** P

Techniques Used: Expressing, Marker, Western Blot, Software

miR-21 regulates EMT through mediating Akt and ERK1/2 activation in QBC939 cells. (A) Relative and (B) semi-quantified protein levels of EMT markers from Blank, NC, miR-21 mimic, LY294002 and U0126 treatment groups. GAPDH was used as loading control ( ** P
Figure Legend Snippet: miR-21 regulates EMT through mediating Akt and ERK1/2 activation in QBC939 cells. (A) Relative and (B) semi-quantified protein levels of EMT markers from Blank, NC, miR-21 mimic, LY294002 and U0126 treatment groups. GAPDH was used as loading control ( ** P

Techniques Used: Activation Assay

miR-21 regulates the invasion and migration of CCA cells through activation of Akt and ERK1/2 pathways. (A) The invasive and (C) migration properties of indicated cells were tested in invasion and migration assays in Transwell inserts. (B and D) Penetrated cells were counted and analyzed. (E) Scratch-wound assay was performed in QBC939 cells and (F) migration rate were quantified from results shown in (E). Experiments were performed in triplicate ( * P
Figure Legend Snippet: miR-21 regulates the invasion and migration of CCA cells through activation of Akt and ERK1/2 pathways. (A) The invasive and (C) migration properties of indicated cells were tested in invasion and migration assays in Transwell inserts. (B and D) Penetrated cells were counted and analyzed. (E) Scratch-wound assay was performed in QBC939 cells and (F) migration rate were quantified from results shown in (E). Experiments were performed in triplicate ( * P

Techniques Used: Migration, Activation Assay, Scratch Wound Assay Assay

30) Product Images from "N-Glycosylation of ss4 Integrin Controls the Adhesion and Motility of Keratinocytes"

Article Title: N-Glycosylation of ss4 Integrin Controls the Adhesion and Motility of Keratinocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027084

Effects of ΔNß4 integrin on cellular signaling. Lac, WT or ΔN keratinocytes were grown to semi-confluence and then each cell lysate was collected as described in “ MATERIALS AND METHODS . ” (A and C) 20 µg of cell lysates from those keratinocytes were run on 7.5% or 5–20% SDS-polyacrylamide gel and probed with a phospho-Akt Ab (A, upper panel) or phospho-ERK1/2 Ab (C, upper panel), and then reprobed with a total Akt Ab (A, lower panel) or total ERK Ab (C, lower panel), respectively. (B and D) Results of the densitometric analysis are shown as the integrated density of the ratio of phospho-protein to total protein bands, which was 1.0 for lac keratinocytes. *p
Figure Legend Snippet: Effects of ΔNß4 integrin on cellular signaling. Lac, WT or ΔN keratinocytes were grown to semi-confluence and then each cell lysate was collected as described in “ MATERIALS AND METHODS . ” (A and C) 20 µg of cell lysates from those keratinocytes were run on 7.5% or 5–20% SDS-polyacrylamide gel and probed with a phospho-Akt Ab (A, upper panel) or phospho-ERK1/2 Ab (C, upper panel), and then reprobed with a total Akt Ab (A, lower panel) or total ERK Ab (C, lower panel), respectively. (B and D) Results of the densitometric analysis are shown as the integrated density of the ratio of phospho-protein to total protein bands, which was 1.0 for lac keratinocytes. *p

Techniques Used:

31) Product Images from "Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics"

Article Title: Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

Journal: Translational Psychiatry

doi: 10.1038/s41398-019-0647-7

Neurotransmitter receptors and associated signalling pathways involved in the control of ADAMTS2 gene expression. a ADAMTS2 mRNA levels by RT-qPCR in SK-N-SH cells incubated 1 h with the indicated selective receptor agonist (red bars): SKF 83822 (D 1 -class receptors) ( N = 5), 7-OH-DPAT (D 2 -class receptors) ( N = 3), 8-OH-DPAT (5-HT 1A receptor) ( N = 3) and TCB-2 (5-HT 2A/2C receptors) ( N = 5); and selective antagonist (blue bars): SCH 39165 (D 1 -class receptors) ( N = 3), L 741,626 (D 2 -class receptors) ( N = 4), WAY 100635 (5-HT 1A receptors) ( N = 3) and MDL 100907 (5-HT 2A receptors) ( N = 3) (Drug concentration 1 µM). b ADAMTS2 mRNA levels by RT-qPCR in cells incubated for 1 h with SKF 83822 ( N = 4) and pre-incubated also for 30 min with SCH 39166 ( N = 4), clozapine ( N = 3), haloperidol ( N = 4), paliperidone ( N = 4) or aripiprazole ( N = 4) (Drug concentration 1 µM). c CREB activity in cells transfected with CRE-Luc alongside pRL-Null: cells were pre-incubated for 1 h with the indicated APDs and then, incubated for 24 h with SKF 82833 (10 µM) ( N = 4). d ADAMTS2 mRNA levels by RT-qPCR: SK-N-SH cells were pre-incubated for 30 min with MAPK/ERK and cAMP-PKA inhibitors (selumetinib 1 µM N = 6 and H89 10 µM N = 4, respectively) and then, incubated for 1 h with SKF 82833 (1 µM) ( N = 5). e CREB activity in cells transfected with CRE-Luc alongside pRL-Null ( N = 4): SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 24 h with SKF 82833 (10 µM). f Western blottings showing relative phosphorylation levels of CREB and ERK1/2: SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 15 min with SKF 82833 (1 µM) ( N = 3). Blots are representative images of each western-blot. Data are mean ± SEM; Student’s t -test: * p
Figure Legend Snippet: Neurotransmitter receptors and associated signalling pathways involved in the control of ADAMTS2 gene expression. a ADAMTS2 mRNA levels by RT-qPCR in SK-N-SH cells incubated 1 h with the indicated selective receptor agonist (red bars): SKF 83822 (D 1 -class receptors) ( N = 5), 7-OH-DPAT (D 2 -class receptors) ( N = 3), 8-OH-DPAT (5-HT 1A receptor) ( N = 3) and TCB-2 (5-HT 2A/2C receptors) ( N = 5); and selective antagonist (blue bars): SCH 39165 (D 1 -class receptors) ( N = 3), L 741,626 (D 2 -class receptors) ( N = 4), WAY 100635 (5-HT 1A receptors) ( N = 3) and MDL 100907 (5-HT 2A receptors) ( N = 3) (Drug concentration 1 µM). b ADAMTS2 mRNA levels by RT-qPCR in cells incubated for 1 h with SKF 83822 ( N = 4) and pre-incubated also for 30 min with SCH 39166 ( N = 4), clozapine ( N = 3), haloperidol ( N = 4), paliperidone ( N = 4) or aripiprazole ( N = 4) (Drug concentration 1 µM). c CREB activity in cells transfected with CRE-Luc alongside pRL-Null: cells were pre-incubated for 1 h with the indicated APDs and then, incubated for 24 h with SKF 82833 (10 µM) ( N = 4). d ADAMTS2 mRNA levels by RT-qPCR: SK-N-SH cells were pre-incubated for 30 min with MAPK/ERK and cAMP-PKA inhibitors (selumetinib 1 µM N = 6 and H89 10 µM N = 4, respectively) and then, incubated for 1 h with SKF 82833 (1 µM) ( N = 5). e CREB activity in cells transfected with CRE-Luc alongside pRL-Null ( N = 4): SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 24 h with SKF 82833 (10 µM). f Western blottings showing relative phosphorylation levels of CREB and ERK1/2: SK-N-SH cells were pre-incubated for 1 h with the indicated inhibitors and then incubated for 15 min with SKF 82833 (1 µM) ( N = 3). Blots are representative images of each western-blot. Data are mean ± SEM; Student’s t -test: * p

Techniques Used: Expressing, Quantitative RT-PCR, Incubation, Concentration Assay, Activity Assay, Transfection, Western Blot

Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D 1 receptors by SKF 83822 (selective D 1 receptor agonist) triggers the expression of ADAMTS2 . Two main pathways seem to be involved: (1) G αs /AC/cAMP/PKA signalling and (2) MEK/ERK1/2 signalling. Downstream of D 1 both PKA and ERK can phosphorylate CREB at Ser133 and activate transcription of ADAMTS2 . Specific activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Specific inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D 1 (dopamine D 1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction.
Figure Legend Snippet: Schematic representation of the mechanisms that control ADAMTS2 gene expression. Selective stimulation of D 1 receptors by SKF 83822 (selective D 1 receptor agonist) triggers the expression of ADAMTS2 . Two main pathways seem to be involved: (1) G αs /AC/cAMP/PKA signalling and (2) MEK/ERK1/2 signalling. Downstream of D 1 both PKA and ERK can phosphorylate CREB at Ser133 and activate transcription of ADAMTS2 . Specific activators of PKA (Forskolin) and MEK (TPA) are highlighted in blue. Specific inhibitors of PKA (H89) and MEK (selumetinib) are coloured in red. DA D 1 (dopamine D 1 receptor), Gsα (G-protein α-subunit), Gβγ (G-protein βγ-subunits), AC (adenyl cyclase), PKA (protein kinase A), CRE (cyclic AMP-responsive element) site and PM (plasmatic membrane). Arrows: direct interaction, dashed arrows: indirect interaction.

Techniques Used: Expressing

32) Product Images from "Bile acids inhibit NAD+-dependent 15-hydroxyprostaglandin dehydrogenase transcription in colonocytes"

Article Title: Bile acids inhibit NAD+-dependent 15-hydroxyprostaglandin dehydrogenase transcription in colonocytes

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00133.2009

CD mediated activation of PKC → ERK1/2 leads to reduced levels of 15-PGDH. A : HCT15 cells were treated with 0–100 μM CD for 10 min. Total PKC activity, cytosolic PKC activity, and membrane PKC activity were measured. Columns, means
Figure Legend Snippet: CD mediated activation of PKC → ERK1/2 leads to reduced levels of 15-PGDH. A : HCT15 cells were treated with 0–100 μM CD for 10 min. Total PKC activity, cytosolic PKC activity, and membrane PKC activity were measured. Columns, means

Techniques Used: Activation Assay, Activity Assay

33) Product Images from "MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib"

Article Title: MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib

Journal: Neuro-Oncology

doi: 10.1093/neuonc/noq017

Sunitinib inhibition of ERK1/2 and Akt phosphorylation is dependent on MGMT status. (A) Phosphorylation of ERK1/2 and Akt-Ser473 in response to sunitinib (SU, 1 µM), RT (4 Gy), and/or TMZ (100 µM) in U87/EV cells and (B) in U87/MGMT cells.
Figure Legend Snippet: Sunitinib inhibition of ERK1/2 and Akt phosphorylation is dependent on MGMT status. (A) Phosphorylation of ERK1/2 and Akt-Ser473 in response to sunitinib (SU, 1 µM), RT (4 Gy), and/or TMZ (100 µM) in U87/EV cells and (B) in U87/MGMT cells.

Techniques Used: Inhibition

34) Product Images from "Atypical activation of the G protein Gαq by the oncogenic mutation Q209P"

Article Title: Atypical activation of the G protein Gαq by the oncogenic mutation Q209P

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005291

Gα q Q209P activates downstream signaling in cells like Gα q Q209L. A , expression of Gα q Q209L or Gα q Q209P leads to similar levels of ERK1/2 phosphorylation. Lysates of HEK293T cells transfected with HA-Gα q (WT or mutants, as indicated) were immunoblotted ( IB ) with the indicated antibodies. One representative result of at least three independent experiments is shown. B , expression of Gα q Q209L or Gα q Q209P leads to similar levels of SRE reporter activation. HEK293T cells were co-transfected with HA-Gα q (WT or mutants, as indicated) and a firefly luciferase reporter driven by the SRE.L promoter and cultured overnight in medium with a low concentration of serum (0.5% FBS) before measuring luminescence. Results are mean ± S.E. ( error bars ) ( n = 4).
Figure Legend Snippet: Gα q Q209P activates downstream signaling in cells like Gα q Q209L. A , expression of Gα q Q209L or Gα q Q209P leads to similar levels of ERK1/2 phosphorylation. Lysates of HEK293T cells transfected with HA-Gα q (WT or mutants, as indicated) were immunoblotted ( IB ) with the indicated antibodies. One representative result of at least three independent experiments is shown. B , expression of Gα q Q209L or Gα q Q209P leads to similar levels of SRE reporter activation. HEK293T cells were co-transfected with HA-Gα q (WT or mutants, as indicated) and a firefly luciferase reporter driven by the SRE.L promoter and cultured overnight in medium with a low concentration of serum (0.5% FBS) before measuring luminescence. Results are mean ± S.E. ( error bars ) ( n = 4).

Techniques Used: Expressing, Transfection, Activation Assay, Luciferase, Cell Culture, Concentration Assay

35) Product Images from "Crucial roles of Nox2-derived oxidative stress in deteriorating the function of insulin receptors and endothelium in dietary obesity of middle-aged mice"

Article Title: Crucial roles of Nox2-derived oxidative stress in deteriorating the function of insulin receptors and endothelium in dietary obesity of middle-aged mice

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12336

Aortic vasomotor response to insulin stimulation and ex vivo organ culture for insulin and high glucose-induced changes in insulin receptor (IR) expression, and ERK1/2 and eNOS phosphorylation. The effect of pre-incubation with insulin (1.2 nM
Figure Legend Snippet: Aortic vasomotor response to insulin stimulation and ex vivo organ culture for insulin and high glucose-induced changes in insulin receptor (IR) expression, and ERK1/2 and eNOS phosphorylation. The effect of pre-incubation with insulin (1.2 nM

Techniques Used: Ex Vivo, Organ Culture, Expressing, Incubation

36) Product Images from "Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts"

Article Title: Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Journal: Biochimie

doi: 10.1016/j.biochi.2011.09.009

Role of C1P-induced activation of Akt, ERK1/2 and mTOR (A) on DNA synthesis (B), and cyclin D1 expression (C) in C2C12 myoblasts. C2C12 myoblasts approximately 40% confluent were serum-starved for 24 h and incubated with or without 15 μM C1P for the indicated time-intervals. A) Cell lysates were separated by SDS-PAGE and immunoblotted using specific anti-phospho-Akt, anti-pan Akt, anti-phospho-ERK1/2, anti-pan ERK1/2, anti-phospho-mTOR and anti-β-actin antibodies. Blots representative of at least three independent experiments are shown. In the histograms band intensity corresponding to phosphorylated protein was normalized to its total content or to β-actin and reported as mean ± SEM of three independent experiments, fold change over control set as 1. The effect of C1P was statistically significant by Student’s t test (* P
Figure Legend Snippet: Role of C1P-induced activation of Akt, ERK1/2 and mTOR (A) on DNA synthesis (B), and cyclin D1 expression (C) in C2C12 myoblasts. C2C12 myoblasts approximately 40% confluent were serum-starved for 24 h and incubated with or without 15 μM C1P for the indicated time-intervals. A) Cell lysates were separated by SDS-PAGE and immunoblotted using specific anti-phospho-Akt, anti-pan Akt, anti-phospho-ERK1/2, anti-pan ERK1/2, anti-phospho-mTOR and anti-β-actin antibodies. Blots representative of at least three independent experiments are shown. In the histograms band intensity corresponding to phosphorylated protein was normalized to its total content or to β-actin and reported as mean ± SEM of three independent experiments, fold change over control set as 1. The effect of C1P was statistically significant by Student’s t test (* P

Techniques Used: Activation Assay, DNA Synthesis, Expressing, Incubation, SDS Page

C1P-induced activation of mTOR is downstream of PI3K/Akt and ERK1/2 signaling pathways in C2C12 myoblasts. C2C12 myoblasts approximately 40% confluent were serum-starved for 24 h and pre-incubated for 30 min with PI3K inhibitor (5 μM LY294002) or MEK inhibitor (5 μM U0126) or PI3Kβ inhibitor (1 μM TGX-221) before being challenged with 15 μM C1P for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted using specific anti-phospho-Akt, anti-pan Akt anti-phospho-ERK1/2, anti-pan ERK1/2, anti-phospho-mTOR and anti-β-actin antibodies. Blots representative of at least three independent experiments are shown. Histograms represent densitometric quantification of phosphorylated protein normalized to its total content or to β-actin and reported as mean ± SEM of three independent experiments, fold change over control set as 1. The effect of C1P was statistically significant by Student’s t test (* P
Figure Legend Snippet: C1P-induced activation of mTOR is downstream of PI3K/Akt and ERK1/2 signaling pathways in C2C12 myoblasts. C2C12 myoblasts approximately 40% confluent were serum-starved for 24 h and pre-incubated for 30 min with PI3K inhibitor (5 μM LY294002) or MEK inhibitor (5 μM U0126) or PI3Kβ inhibitor (1 μM TGX-221) before being challenged with 15 μM C1P for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted using specific anti-phospho-Akt, anti-pan Akt anti-phospho-ERK1/2, anti-pan ERK1/2, anti-phospho-mTOR and anti-β-actin antibodies. Blots representative of at least three independent experiments are shown. Histograms represent densitometric quantification of phosphorylated protein normalized to its total content or to β-actin and reported as mean ± SEM of three independent experiments, fold change over control set as 1. The effect of C1P was statistically significant by Student’s t test (* P

Techniques Used: Activation Assay, Incubation, SDS Page

37) Product Images from "Activation of Peroxisome Proliferator-Activated Receptor-?/-? (PPAR-?/-?) Ameliorates Insulin Signaling and Reduces SOCS3 Levels by Inhibiting STAT3 in Interleukin-6-Stimulated Adipocytes"

Article Title: Activation of Peroxisome Proliferator-Activated Receptor-?/-? (PPAR-?/-?) Ameliorates Insulin Signaling and Reduces SOCS3 Levels by Inhibiting STAT3 in Interleukin-6-Stimulated Adipocytes

Journal: Diabetes

doi: 10.2337/db10-0704

The PPAR-δ agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in 3T3-L1 adipocytes. A : Analysis of the mRNA levels of Socs3 in serum-starved differentiated adipocytes untreated or treated with 10 μmol/L GW501516 for 24 h or 2 μmol/L geldanamycine for 30 min before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the Aprt mRNA levels are shown. Data are the means ± SD of five independent experiments. 3T3-L1 adipocytes were treated with 10 μmol/L GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 24 h. Nuclear ( B ) or total cell extracts ( C ) were subjected to Western blot analysis with phospho-STAT3 (Tyr 705 and Ser 727 ) or STAT3 antibodies ( B ) or phospho-ERK1/2 and ERK1/2 ( C ) antibodies. D : Analysis of the mRNA levels of Egr-1 in 3T3-L1 serum-starved differentiated adipocytes untreated or treated with 10 μmol/L GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. Bars are the means ± SD of five independent experiments. * P
Figure Legend Snippet: The PPAR-δ agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in 3T3-L1 adipocytes. A : Analysis of the mRNA levels of Socs3 in serum-starved differentiated adipocytes untreated or treated with 10 μmol/L GW501516 for 24 h or 2 μmol/L geldanamycine for 30 min before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the Aprt mRNA levels are shown. Data are the means ± SD of five independent experiments. 3T3-L1 adipocytes were treated with 10 μmol/L GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 24 h. Nuclear ( B ) or total cell extracts ( C ) were subjected to Western blot analysis with phospho-STAT3 (Tyr 705 and Ser 727 ) or STAT3 antibodies ( B ) or phospho-ERK1/2 and ERK1/2 ( C ) antibodies. D : Analysis of the mRNA levels of Egr-1 in 3T3-L1 serum-starved differentiated adipocytes untreated or treated with 10 μmol/L GW501516 for 24 h before stimulation with 10 ng/mL IL-6 for 1 h. Total RNA was isolated and analyzed by RT-PCR. Bars are the means ± SD of five independent experiments. * P

Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

ERK1/2 inhibition prevents IL-6–induced insulin resistance and STAT3 phosphorylation on Ser 727 . Differentiated adipocytes were stimulated with 100 nmol/L insulin for 30 min, with or without pretreatment with either 10 μmol/L U0126, 10 μmol/L GW501516, or 100 ng/mL IL-6 for 24 h. A : Cell lysates were subjected to Western blot analysis for phospho-Akt(Ser 473 ) and total Akt and β-actin. B : 2-Deoxyglucose uptake was assessed without or with insulin. Values are means ± SD of six independent experiments. C : Nuclear cell extracts were subjected to Western blot analysis with phospho-STAT3 (Ser 727 ), STAT3, or Lamin B antibodies. *** P
Figure Legend Snippet: ERK1/2 inhibition prevents IL-6–induced insulin resistance and STAT3 phosphorylation on Ser 727 . Differentiated adipocytes were stimulated with 100 nmol/L insulin for 30 min, with or without pretreatment with either 10 μmol/L U0126, 10 μmol/L GW501516, or 100 ng/mL IL-6 for 24 h. A : Cell lysates were subjected to Western blot analysis for phospho-Akt(Ser 473 ) and total Akt and β-actin. B : 2-Deoxyglucose uptake was assessed without or with insulin. Values are means ± SD of six independent experiments. C : Nuclear cell extracts were subjected to Western blot analysis with phospho-STAT3 (Ser 727 ), STAT3, or Lamin B antibodies. *** P

Techniques Used: Inhibition, Western Blot

The PPAR-δ agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in white adipose tissue. Phospho-STAT3 (Ser 727 ) and SOCS3 protein levels are increased in white adipose tissue of ZDF rats. A : Analysis of phospho-STAT3 (Ser 727 ) and total STAT3 by immunoblotting of nuclear or total protein extracts from white adipose tissue of lean and ZDF rats. B : Total cell extracts from white adipose tissue of lean and ZDF rats were subjected to Western blot analysis with SOCS3 and β-actin antibodies. Mice were treated for 48 h with vehicle, IL-6, or IL-6 plus GW501516. SOCS3 ( C ), phospho-STAT3 (Tyr 705 and Ser 727 ) ( D ), and phospho-ERK1/2 ( E ) protein levels. Nuclear (phospho-STAT3-Ser 727 ) or total cell extracts were subjected to Western blot analysis with phospho-STAT3 (Tyr 705 ) or STAT3 antibodies or phospho-ERK1/2 and ERK1/2 antibodies. Bars are the means ± SD of four independent experiments. *** P
Figure Legend Snippet: The PPAR-δ agonist GW501516 prevents IL-6–induced SOCS3 expression and STAT3 phosphorylation in white adipose tissue. Phospho-STAT3 (Ser 727 ) and SOCS3 protein levels are increased in white adipose tissue of ZDF rats. A : Analysis of phospho-STAT3 (Ser 727 ) and total STAT3 by immunoblotting of nuclear or total protein extracts from white adipose tissue of lean and ZDF rats. B : Total cell extracts from white adipose tissue of lean and ZDF rats were subjected to Western blot analysis with SOCS3 and β-actin antibodies. Mice were treated for 48 h with vehicle, IL-6, or IL-6 plus GW501516. SOCS3 ( C ), phospho-STAT3 (Tyr 705 and Ser 727 ) ( D ), and phospho-ERK1/2 ( E ) protein levels. Nuclear (phospho-STAT3-Ser 727 ) or total cell extracts were subjected to Western blot analysis with phospho-STAT3 (Tyr 705 ) or STAT3 antibodies or phospho-ERK1/2 and ERK1/2 antibodies. Bars are the means ± SD of four independent experiments. *** P

Techniques Used: Expressing, Western Blot, Mouse Assay

38) Product Images from "Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells"

Article Title: Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044107

Inhibition of the phosphorylation of CREB, and MAPKs signaling by resveratrol is PI3-K-dependent during RAW 264.7 macrophage cells activation by LPS. Approximately 1×10 6 cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (1, 5, and 10 µM) for 1 h in the absence or presence of wortmannin (1 µM), then exposed to LPS (5 µg/ml) for 30 min. Cell lysates (50 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for phosphorylated forms of CREB, ERK1/2, JNK and p38 MAPK (shown as phospho-IκB-α, etc.) as described in the methods . Equivalent loading of cell lysates was determined by reprobing the blots with anti-β-actin, total ERK1/2, JNK or p38 MAPK antibodies. The relative protein levels were quantified by scanning densitometry and normalized to β-actin, total ERK1/2, JNK or p38 MAPK. The values shown are mean ± SEM of data from three independent experiments. # Significant compared with control alone, p
Figure Legend Snippet: Inhibition of the phosphorylation of CREB, and MAPKs signaling by resveratrol is PI3-K-dependent during RAW 264.7 macrophage cells activation by LPS. Approximately 1×10 6 cells/ml were seeded in six-well plates and incubated until 80% confluency. Cells were pre-treated with resveratrol (1, 5, and 10 µM) for 1 h in the absence or presence of wortmannin (1 µM), then exposed to LPS (5 µg/ml) for 30 min. Cell lysates (50 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for phosphorylated forms of CREB, ERK1/2, JNK and p38 MAPK (shown as phospho-IκB-α, etc.) as described in the methods . Equivalent loading of cell lysates was determined by reprobing the blots with anti-β-actin, total ERK1/2, JNK or p38 MAPK antibodies. The relative protein levels were quantified by scanning densitometry and normalized to β-actin, total ERK1/2, JNK or p38 MAPK. The values shown are mean ± SEM of data from three independent experiments. # Significant compared with control alone, p

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

39) Product Images from "Elevated expression of syntenin in breast cancer is correlated with lymph node metastasis and poor patient survival"

Article Title: Elevated expression of syntenin in breast cancer is correlated with lymph node metastasis and poor patient survival

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3442

Activation of integrin β1 and ERK1/2 is essential for syntenin-induced migration and invasion . (A) Silencing of syntenin in MDA-MB-231HM cells inhibited active integrin β1 expression and phosphorylation of ERK1/2, but had no effects on JNK and p38. (B) Overexpression of syntenin increased active integrin β1 expression and ERK1/2 phosphorylation in 231-SYN cells. The activation of ERK1/2 was analyzed with Western blot by using phosphor-specific antibodies. (C) Integrin β1 functional blocking antibody blocked both active integrin β1 expression and ERK1/2 phosphorylation. Cells were treated with integrin β1 or nonspecific IgG for 1 hour before protein extraction. (D) ERK1/2 inhibitor U0126 blocked activation of ERK1/2. Cells were pretreated with dimethylsulfoxide (DMSO) or 20 µ M U0126 (U0126) for 2 hours before protein extraction. (E, F) U0126 effectively reduced the migration and invasion of breast cancer cells. GAPDH was used as loading control. Data are expressed as means of triplicate samples from three independent experiments; bars, SD. ** P
Figure Legend Snippet: Activation of integrin β1 and ERK1/2 is essential for syntenin-induced migration and invasion . (A) Silencing of syntenin in MDA-MB-231HM cells inhibited active integrin β1 expression and phosphorylation of ERK1/2, but had no effects on JNK and p38. (B) Overexpression of syntenin increased active integrin β1 expression and ERK1/2 phosphorylation in 231-SYN cells. The activation of ERK1/2 was analyzed with Western blot by using phosphor-specific antibodies. (C) Integrin β1 functional blocking antibody blocked both active integrin β1 expression and ERK1/2 phosphorylation. Cells were treated with integrin β1 or nonspecific IgG for 1 hour before protein extraction. (D) ERK1/2 inhibitor U0126 blocked activation of ERK1/2. Cells were pretreated with dimethylsulfoxide (DMSO) or 20 µ M U0126 (U0126) for 2 hours before protein extraction. (E, F) U0126 effectively reduced the migration and invasion of breast cancer cells. GAPDH was used as loading control. Data are expressed as means of triplicate samples from three independent experiments; bars, SD. ** P

Techniques Used: Activation Assay, Migration, Multiple Displacement Amplification, Expressing, Over Expression, Western Blot, Functional Assay, Blocking Assay, Protein Extraction

40) Product Images from "Angiotensin II produces nociceptive behavior through spinal AT1 receptor-mediated p38 mitogen-activated protein kinase activation in mice"

Article Title: Angiotensin II produces nociceptive behavior through spinal AT1 receptor-mediated p38 mitogen-activated protein kinase activation in mice

Journal: Molecular Pain

doi: 10.1186/1744-8069-9-38

Alterations in spinal MAPKs phosphorylation by Ang II and the effects of losartan and PD123319. Dorsal spinal cord samples were taken 10 min after i.t. injection of Ang II (3 pmol). Phosphorylation of ERK1/2 (a) , JNK (b) and p38 MAPK (c , d) were examined by Western blotting. Losartan (3 nmol) or PD123319 (3 nmol) was co-administered i.t. with Ang II. Top: representative Western blot of total- and phospho-MAPKs. Bottom: quantification of phospho-MAPKs to total-MAPKs set as 1.0 in the Ringer- or vehicle (6.8% DMSO)-treated group. Each value represents the means ± S.E.M. of 4 mice in each group. * p
Figure Legend Snippet: Alterations in spinal MAPKs phosphorylation by Ang II and the effects of losartan and PD123319. Dorsal spinal cord samples were taken 10 min after i.t. injection of Ang II (3 pmol). Phosphorylation of ERK1/2 (a) , JNK (b) and p38 MAPK (c , d) were examined by Western blotting. Losartan (3 nmol) or PD123319 (3 nmol) was co-administered i.t. with Ang II. Top: representative Western blot of total- and phospho-MAPKs. Bottom: quantification of phospho-MAPKs to total-MAPKs set as 1.0 in the Ringer- or vehicle (6.8% DMSO)-treated group. Each value represents the means ± S.E.M. of 4 mice in each group. * p

Techniques Used: Injection, Western Blot, Mouse Assay

Related Articles

Western Blot:

Article Title: Coordinated Regulation of Nrf2 and Histone H3 Serine 10 Phosphorylation in Arsenite-activated Transcription of the Human Heme Oxygenase-1 Gene
Article Snippet: .. Antibodies utilized in Western blotting and/or chromatin immunoprecipitation assay (ChIP) were purchased from the following companies: anti-heme oxygenase-1 (sc-7695), anti-Nrf2 (sc-13032X), anti-JNK (sc-571), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025), and anti-RNAPII (sc-899X) all from Santa Cruz Biotechnology; anti-lamin B (Ab-1) from Oncogene; anti-lactate dehydrogenase (AB1222) from Chemicon; phospho-specific antibodies of p38 MAPK (9211), MEK1/2 (9121), JNK (4688), ERK1/2 (9911), histone H3S10 (3377), and anti-Histone H3 (9715) all from Cell Signaling Technology; ChIP grade phospho-specific anti-H3S10 (ab14955), anti-RNAPIISer2 (ab5095), and anti-RNAPIISer5 (ab5131) from Abcam; and anti-β-actin (A5441) from Sigma. .. Whole cell extracts (WCE) were prepared by lysing cells with lysis buffer (150mM NaCl, 10mM Na2 HPO4 , 1% Triton-X, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), and 0.2% sodium azide; pH 7.4).

Incubation:

Article Title: MicroRNA-211 loss promotes metabolic vulnerability and BRAF inhibitor sensitivity in melanoma
Article Snippet: .. After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with antibodies against phospho-ERK1/2 (Cell signaling, Phospho-Erk1/2 Pathway Sampler Kit #9911); phospho-PTEN and phospho-AKT, total AKT (Cell signaling, Phospho-Akt Pathway Antibody Sampler Kit #9916), total ERK, total PTEN, phospho-mTOR, total mTOR (Cell signaling) OXPHOS (Cat. No. ab 110413) and GAPDH (Santa Cruz Biotechnology)at 4 °C for 12 h. Membranes were washed three times for 10 min and incubated with a 1:3000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 h. Blots were washed with TBST three times and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocols. .. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured as described previously ( ) using the XF Extracellular Flux Analyzer (Seahorse Bioscience, Agilent) and Seahorse XF Cell Mito Stress Test Kit (#103015-100; Seahorse Bioscience, Agilent) with minor modifications to the manufacturer’s protocols.

Chromatin Immunoprecipitation:

Article Title: Coordinated Regulation of Nrf2 and Histone H3 Serine 10 Phosphorylation in Arsenite-activated Transcription of the Human Heme Oxygenase-1 Gene
Article Snippet: .. Antibodies utilized in Western blotting and/or chromatin immunoprecipitation assay (ChIP) were purchased from the following companies: anti-heme oxygenase-1 (sc-7695), anti-Nrf2 (sc-13032X), anti-JNK (sc-571), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025), and anti-RNAPII (sc-899X) all from Santa Cruz Biotechnology; anti-lamin B (Ab-1) from Oncogene; anti-lactate dehydrogenase (AB1222) from Chemicon; phospho-specific antibodies of p38 MAPK (9211), MEK1/2 (9121), JNK (4688), ERK1/2 (9911), histone H3S10 (3377), and anti-Histone H3 (9715) all from Cell Signaling Technology; ChIP grade phospho-specific anti-H3S10 (ab14955), anti-RNAPIISer2 (ab5095), and anti-RNAPIISer5 (ab5131) from Abcam; and anti-β-actin (A5441) from Sigma. .. Whole cell extracts (WCE) were prepared by lysing cells with lysis buffer (150mM NaCl, 10mM Na2 HPO4 , 1% Triton-X, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), and 0.2% sodium azide; pH 7.4).

Activity Assay:

Article Title: Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1
Article Snippet: .. For analysis of ERK/MAPK signaling, the phospho-ERK1/2 pathway kit (9911, Cell Signaling) was used for MEK/ERK/P90RSK ; Raf-1 (9422, Cell Signaling); phosho-(S338)-Raf-1 (9924, Cell Signaling); B-Raf (mAb L12G7, Cell signaling, 9434); phosphor-(Ser445)-B-Raf (2696, Cell Signaling); Ras activity assay kit (17–218, Upstate). ..

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    Cell Signaling Technology Inc erk1 2
    Hon inhibits IFNγ-induced activation of <t>p-ERK1/2</t> 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
    Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 3087 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/erk1 2/product/Cell Signaling Technology Inc
    Average 95 stars, based on 3087 article reviews
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    erk1 2 - by Bioz Stars, 2020-09
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    85
    Cell Signaling Technology Inc reagents phospho erk1 2 perk1 2 rabbit monoclonal antibody
    Glutamate-dependent activation of <t>Erk1/2</t> is blocked by B355252 in HT-22 cells . Cultured cells were treated with glutamate 10 h following a 2 h pretreatment with B355252. Cellular fractions (cytosolic and nuclear) were prepared and subjected to immunoblot analysis with total Erk1/2 or <t>pERK1/2</t> specific antibodies. Membranes were reprobed with GAPDH and H3 histone specific antibodies as controls for protein loading. (A) . Cytoplasmic fraction probed with total Erk1/2 (top), pERK1/2 (middle), and GAPDH (bottom) specific antibodies. Erk activation by glutamate was significantly attenuated by pretreatment of cells with B355252 (8 μM). B355252 alone had no effect on phospho-Erk1/2. The bar graph in figure A shows the relative level of active Erk in the samples. ***p
    Reagents Phospho Erk1 2 Perk1 2 Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reagents phospho erk1 2 perk1 2 rabbit monoclonal antibody - by Bioz Stars, 2020-09
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    Cell Signaling Technology Inc monoclonal anti phospho erk1 2 antibody
    A set of histograms showing in situ hybridization of erk2 in VSMCs of small renal arteries. Little to none erk2 mRNA expression (▴) was observed in WKY rats at 16 and 24 weeks ( A , D ). Marked increase of erk2 mRNA expression (↑) was detected in SHR at 16 and 24 weeks of age group ( B , E ). Erk2 mRNA was reduced in PD98059-treaetd SHR (↑) at 16 and 24 weeks of age group( C , F ). Arrow heads denote negative cells and arrows denote positive cells. DAB was used as substrate. Sections were counterstained with hematoxylin. Magnification, 400×. ( G ) summarized bar graph showing number of in situ <t>phospho-ERK1/2</t> VSMCs over total observed VSMCs in three groups of animals at 16 and 24 weeks. * P
    Monoclonal Anti Phospho Erk1 2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti phospho erk1 2 antibody/product/Cell Signaling Technology Inc
    Average 88 stars, based on 3 article reviews
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    Image Search Results


    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

    Journal: Journal of Neuroinflammation

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

    doi: 10.1186/1742-2094-10-15

    Figure Lengend 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

    Article Snippet: Our results here also demonstrated the ability for IFNγ alone to stimulate phosphorylation of ERK1/2 in microglial cells (Figure A, B).

    Techniques: 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).

    Journal: Journal of Neuroinflammation

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

    doi: 10.1186/1742-2094-10-15

    Figure Lengend 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).

    Article Snippet: Our results here also demonstrated the ability for IFNγ alone to stimulate phosphorylation of ERK1/2 in microglial cells (Figure A, B).

    Techniques: 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

    Journal: Journal of Neuroinflammation

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

    doi: 10.1186/1742-2094-10-15

    Figure Lengend 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

    Article Snippet: Our results here also demonstrated the ability for IFNγ alone to stimulate phosphorylation of ERK1/2 in microglial cells (Figure A, B).

    Techniques: 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

    Journal: Journal of Neuroinflammation

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

    doi: 10.1186/1742-2094-10-15

    Figure Lengend 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

    Article Snippet: Our results here also demonstrated the ability for IFNγ alone to stimulate phosphorylation of ERK1/2 in microglial cells (Figure A, B).

    Techniques: Activation Assay, Western Blot

    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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2014/253075

    Figure Lengend 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

    Article Snippet: In a middle cerebral artery occlusion (MCAO) model, EA at either GV20 (Baihui) or DU26 (Renzhong) acupoints increases the phosphorylation of ERK1/2 in the ischemic cortex and hippocampus [ ].

    Techniques: 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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2014/253075

    Figure Lengend 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

    Article Snippet: In a middle cerebral artery occlusion (MCAO) model, EA at either GV20 (Baihui) or DU26 (Renzhong) acupoints increases the phosphorylation of ERK1/2 in the ischemic cortex and hippocampus [ ].

    Techniques: 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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2014/253075

    Figure Lengend 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

    Article Snippet: In a middle cerebral artery occlusion (MCAO) model, EA at either GV20 (Baihui) or DU26 (Renzhong) acupoints increases the phosphorylation of ERK1/2 in the ischemic cortex and hippocampus [ ].

    Techniques: 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

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2014/253075

    Figure Lengend 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

    Article Snippet: In a middle cerebral artery occlusion (MCAO) model, EA at either GV20 (Baihui) or DU26 (Renzhong) acupoints increases the phosphorylation of ERK1/2 in the ischemic cortex and hippocampus [ ].

    Techniques: 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.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

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

    doi: 10.1155/2014/253075

    Figure Lengend 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.

    Article Snippet: In a middle cerebral artery occlusion (MCAO) model, EA at either GV20 (Baihui) or DU26 (Renzhong) acupoints increases the phosphorylation of ERK1/2 in the ischemic cortex and hippocampus [ ].

    Techniques: Western Blot

    Glutamate-dependent activation of Erk1/2 is blocked by B355252 in HT-22 cells . Cultured cells were treated with glutamate 10 h following a 2 h pretreatment with B355252. Cellular fractions (cytosolic and nuclear) were prepared and subjected to immunoblot analysis with total Erk1/2 or pERK1/2 specific antibodies. Membranes were reprobed with GAPDH and H3 histone specific antibodies as controls for protein loading. (A) . Cytoplasmic fraction probed with total Erk1/2 (top), pERK1/2 (middle), and GAPDH (bottom) specific antibodies. Erk activation by glutamate was significantly attenuated by pretreatment of cells with B355252 (8 μM). B355252 alone had no effect on phospho-Erk1/2. The bar graph in figure A shows the relative level of active Erk in the samples. ***p

    Journal: BMC Neuroscience

    Article Title: A novel phenoxy thiophene sulphonamide molecule protects against glutamate evoked oxidative injury in a neuronal cell model

    doi: 10.1186/1471-2202-14-93

    Figure Lengend Snippet: Glutamate-dependent activation of Erk1/2 is blocked by B355252 in HT-22 cells . Cultured cells were treated with glutamate 10 h following a 2 h pretreatment with B355252. Cellular fractions (cytosolic and nuclear) were prepared and subjected to immunoblot analysis with total Erk1/2 or pERK1/2 specific antibodies. Membranes were reprobed with GAPDH and H3 histone specific antibodies as controls for protein loading. (A) . Cytoplasmic fraction probed with total Erk1/2 (top), pERK1/2 (middle), and GAPDH (bottom) specific antibodies. Erk activation by glutamate was significantly attenuated by pretreatment of cells with B355252 (8 μM). B355252 alone had no effect on phospho-Erk1/2. The bar graph in figure A shows the relative level of active Erk in the samples. ***p

    Article Snippet: Antibodies and reagents Phospho-Erk1/2 (pERK1/2) rabbit monoclonal antibody, ERK3, and histone H3 rabbit antibody were purchased from Cell Signaling Technology (Danvers, MA) and Epitomics, Inc (Burlingame, CA).

    Techniques: Activation Assay, Cell Culture

    A set of histograms showing in situ hybridization of erk2 in VSMCs of small renal arteries. Little to none erk2 mRNA expression (▴) was observed in WKY rats at 16 and 24 weeks ( A , D ). Marked increase of erk2 mRNA expression (↑) was detected in SHR at 16 and 24 weeks of age group ( B , E ). Erk2 mRNA was reduced in PD98059-treaetd SHR (↑) at 16 and 24 weeks of age group( C , F ). Arrow heads denote negative cells and arrows denote positive cells. DAB was used as substrate. Sections were counterstained with hematoxylin. Magnification, 400×. ( G ) summarized bar graph showing number of in situ phospho-ERK1/2 VSMCs over total observed VSMCs in three groups of animals at 16 and 24 weeks. * P

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Extracellular Signal-Regulated Kinases Ameliorates Hypertension-Induced Renal Vascular Remodeling in Rat Models

    doi: 10.3390/ijms12128333

    Figure Lengend Snippet: A set of histograms showing in situ hybridization of erk2 in VSMCs of small renal arteries. Little to none erk2 mRNA expression (▴) was observed in WKY rats at 16 and 24 weeks ( A , D ). Marked increase of erk2 mRNA expression (↑) was detected in SHR at 16 and 24 weeks of age group ( B , E ). Erk2 mRNA was reduced in PD98059-treaetd SHR (↑) at 16 and 24 weeks of age group( C , F ). Arrow heads denote negative cells and arrows denote positive cells. DAB was used as substrate. Sections were counterstained with hematoxylin. Magnification, 400×. ( G ) summarized bar graph showing number of in situ phospho-ERK1/2 VSMCs over total observed VSMCs in three groups of animals at 16 and 24 weeks. * P

    Article Snippet: Monoclonal anti-phospho-ERK1/2 antibody (Cell Signaling), ERK1/2 inhibitor PD98059 (Merck), Streptavidinbiotin complex kits (Zymed), and protein markers were purchased from Wuhan Boster Biological Engineering Limited (Wuhan, China).

    Techniques: In Situ Hybridization, Expressing, In Situ

    Phospho-ERK1/2 immunohistochemistry in endothelial cells of the small renal arteries (A–F). ( A ) no expression of phospho-ERK1/2 in WKY rats at 16 weeks of age group (▴); ( B ) increase in number of phospho-ERK1/2 positive cells in SHR at 16 weeks of age group (↑); ( C ) reduction in number of phospho-ERK1/2 positive cells in PD98059-treated SHR at 16 weeks of age group (↑); ( D ) no expression of phospho-ERK1/2 in WKY rats at 24 weeks of age group (▴); ( E ) increase in number of phospho-ERK1/2 positive cells in SHR at 24 weeks of age group; ( F ) significantly decrease in number of phospho-ERK1/2 positive cells in PD98059-treated SHR at 24 weeks of aged group (↑). DAB was used as substrate. Hematoxylin counterstaining. Magnification, 400×. ( G ) summarized data showing number of phospho-ERK1/2 positive endothelial cells of the small renal arteries. * P

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Extracellular Signal-Regulated Kinases Ameliorates Hypertension-Induced Renal Vascular Remodeling in Rat Models

    doi: 10.3390/ijms12128333

    Figure Lengend Snippet: Phospho-ERK1/2 immunohistochemistry in endothelial cells of the small renal arteries (A–F). ( A ) no expression of phospho-ERK1/2 in WKY rats at 16 weeks of age group (▴); ( B ) increase in number of phospho-ERK1/2 positive cells in SHR at 16 weeks of age group (↑); ( C ) reduction in number of phospho-ERK1/2 positive cells in PD98059-treated SHR at 16 weeks of age group (↑); ( D ) no expression of phospho-ERK1/2 in WKY rats at 24 weeks of age group (▴); ( E ) increase in number of phospho-ERK1/2 positive cells in SHR at 24 weeks of age group; ( F ) significantly decrease in number of phospho-ERK1/2 positive cells in PD98059-treated SHR at 24 weeks of aged group (↑). DAB was used as substrate. Hematoxylin counterstaining. Magnification, 400×. ( G ) summarized data showing number of phospho-ERK1/2 positive endothelial cells of the small renal arteries. * P

    Article Snippet: Monoclonal anti-phospho-ERK1/2 antibody (Cell Signaling), ERK1/2 inhibitor PD98059 (Merck), Streptavidinbiotin complex kits (Zymed), and protein markers were purchased from Wuhan Boster Biological Engineering Limited (Wuhan, China).

    Techniques: Immunohistochemistry, Expressing

    Phospho-ERK1/2 immunohistochemistry in VSMCs of interlobar arterioles. A – F , representative ERK1/2 immunohistostaining in VSMCs. ( A ) no expression of phospho-ERK1/2 in WKY at 16 weeks of age group (▴); ( B ) increased expression of phospho-ERK1/2 in SHR at 16 weeks of age group (↑); ( C) reduced expression of phospho-ERK1/2 in PD98059-treated SHR at 16 weeks of age group (↑); ( D) no expression of phospho-ERK1/2 in WKY rats at 24 weeks of age group (▴); ( E) increased expression of phospho-ERK1/2 in SHR at 24 weeks of age group; ( F ) reduced expression of phospho-ERK1/2 VSMC except distal renal tubule (↑) in PD98059-treated SHR at 24 weeks of age group (▴). DAB visualized, slight hematoxylin counterstaining. Magnification, 400×. G , Summary of phospho-ERK1/2 positively stained VSMCs in the small renal arteries. * P

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Extracellular Signal-Regulated Kinases Ameliorates Hypertension-Induced Renal Vascular Remodeling in Rat Models

    doi: 10.3390/ijms12128333

    Figure Lengend Snippet: Phospho-ERK1/2 immunohistochemistry in VSMCs of interlobar arterioles. A – F , representative ERK1/2 immunohistostaining in VSMCs. ( A ) no expression of phospho-ERK1/2 in WKY at 16 weeks of age group (▴); ( B ) increased expression of phospho-ERK1/2 in SHR at 16 weeks of age group (↑); ( C) reduced expression of phospho-ERK1/2 in PD98059-treated SHR at 16 weeks of age group (↑); ( D) no expression of phospho-ERK1/2 in WKY rats at 24 weeks of age group (▴); ( E) increased expression of phospho-ERK1/2 in SHR at 24 weeks of age group; ( F ) reduced expression of phospho-ERK1/2 VSMC except distal renal tubule (↑) in PD98059-treated SHR at 24 weeks of age group (▴). DAB visualized, slight hematoxylin counterstaining. Magnification, 400×. G , Summary of phospho-ERK1/2 positively stained VSMCs in the small renal arteries. * P

    Article Snippet: Monoclonal anti-phospho-ERK1/2 antibody (Cell Signaling), ERK1/2 inhibitor PD98059 (Merck), Streptavidinbiotin complex kits (Zymed), and protein markers were purchased from Wuhan Boster Biological Engineering Limited (Wuhan, China).

    Techniques: Immunohistochemistry, Expressing, Staining