Structured Review

Santa Cruz Biotechnology anti erk
(a–c) Effects of cyclic strain on MAPK phosphorylation. Representative Western blots of phosphospecific and total <t>ERK</t> (a), JNK (b), and <t>p38</t> (c) from cell lysates collected at the indicated times after cyclic strain treatment. Optical density measurements were obtained to determine the relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (mean ± SEM, n = 4) indicate the fold change in phosphorylation relative to static controls for each individual experiment. ∗ indicates a significant difference from the static control ( P
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1) Product Images from "Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells"

Article Title: Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2018/7861518

(a–c) Effects of cyclic strain on MAPK phosphorylation. Representative Western blots of phosphospecific and total ERK (a), JNK (b), and p38 (c) from cell lysates collected at the indicated times after cyclic strain treatment. Optical density measurements were obtained to determine the relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (mean ± SEM, n = 4) indicate the fold change in phosphorylation relative to static controls for each individual experiment. ∗ indicates a significant difference from the static control ( P
Figure Legend Snippet: (a–c) Effects of cyclic strain on MAPK phosphorylation. Representative Western blots of phosphospecific and total ERK (a), JNK (b), and p38 (c) from cell lysates collected at the indicated times after cyclic strain treatment. Optical density measurements were obtained to determine the relative amounts of phosphorylated MAPK normalized by the respective total MAPK. The values (mean ± SEM, n = 4) indicate the fold change in phosphorylation relative to static controls for each individual experiment. ∗ indicates a significant difference from the static control ( P

Techniques Used: Western Blot

2) Product Images from "The Active Form of Vitamin D Transcriptionally Represses Smad7 Signaling and Activates Extracellular Signal-regulated Kinase (ERK) to Inhibit the Differentiation of a Inflammatory T Helper Cell Subset and Suppress Experimental Autoimmune Encephalomyelitis *"

Article Title: The Active Form of Vitamin D Transcriptionally Represses Smad7 Signaling and Activates Extracellular Signal-regulated Kinase (ERK) to Inhibit the Differentiation of a Inflammatory T Helper Cell Subset and Suppress Experimental Autoimmune Encephalomyelitis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.621839

A schematic representation of the 1,25(OH) 2 D 3 -VDR axis modulation of Smad and non-Smad ERK pathways in Th17 cells. 1,25(OH) 2 D 3 -bound VDR forms a complex with RXR. Activated Smad3 also interacts with VDR-RXR heterodimer. This complex enters the nucleus and is recruited on to Smad7 promoter. Later, this complex recruits co-repressor HDAC2, and this repressive complex represses the Smad7 expression. On the other hand, 1,25(OH) 2 D 3 -VDR also activates ERK. A decrease in Smad7 expression would contribute to the increased expression of Smad3. 1,25(OH) 2 D 3 -activated ERK may also contribute to the expression and activation of Smad3, which in turn promotes the VDR function by physically interacting with VDR. 1,25(OH) 2 D 3 abrogates the Th17 phenotype by down-regulating IL17, IL23R, IL22, and Smad7 and up-regulating IL10, VDR, Smad3, and activation of ERK.
Figure Legend Snippet: A schematic representation of the 1,25(OH) 2 D 3 -VDR axis modulation of Smad and non-Smad ERK pathways in Th17 cells. 1,25(OH) 2 D 3 -bound VDR forms a complex with RXR. Activated Smad3 also interacts with VDR-RXR heterodimer. This complex enters the nucleus and is recruited on to Smad7 promoter. Later, this complex recruits co-repressor HDAC2, and this repressive complex represses the Smad7 expression. On the other hand, 1,25(OH) 2 D 3 -VDR also activates ERK. A decrease in Smad7 expression would contribute to the increased expression of Smad3. 1,25(OH) 2 D 3 -activated ERK may also contribute to the expression and activation of Smad3, which in turn promotes the VDR function by physically interacting with VDR. 1,25(OH) 2 D 3 abrogates the Th17 phenotype by down-regulating IL17, IL23R, IL22, and Smad7 and up-regulating IL10, VDR, Smad3, and activation of ERK.

Techniques Used: Expressing, Activation Assay

1,25(OH) 2 D 3 abrogates Smad7 expression and activates ERK in EAE mice. A , C57BL/6 mice were immunized with MOG, and clinical scores were monitored and expressed as the mean ± S.D. B , at day 17 (peak of the disease), total CD4 cells from spleen and lymph nodes were isolated, and the expression of Smad3, p-Smad3, and Smad7 was analyzed by immunoblot analysis. Quantified data were shown above the each panel. C , ChIP experiment performed in CD4 cells isolated from untreated and EAE and EAE mice treated with 1,25(OH) 2 D 3 . ChIP/re-ChIP was performed with antibodies specific for VDR, RXRα, p-Smad3, and HDAC2. D , immunoblot analysis of p-ERK and ERK from total CD4 cells isolated from spleen and lymph nodes of unimmunized and EAE and EAE mice treated with 1,25(OH) 2 D 3. shown is a quantitative representation of blots by normalizing p-ERK protein level over ERK protein level. Data are representative of three independent experiments with at least five mice for each group. In A and C , data represent the mean ± S.D. ( error bars ) of three independent experiments each performed in triplicate. *, p
Figure Legend Snippet: 1,25(OH) 2 D 3 abrogates Smad7 expression and activates ERK in EAE mice. A , C57BL/6 mice were immunized with MOG, and clinical scores were monitored and expressed as the mean ± S.D. B , at day 17 (peak of the disease), total CD4 cells from spleen and lymph nodes were isolated, and the expression of Smad3, p-Smad3, and Smad7 was analyzed by immunoblot analysis. Quantified data were shown above the each panel. C , ChIP experiment performed in CD4 cells isolated from untreated and EAE and EAE mice treated with 1,25(OH) 2 D 3 . ChIP/re-ChIP was performed with antibodies specific for VDR, RXRα, p-Smad3, and HDAC2. D , immunoblot analysis of p-ERK and ERK from total CD4 cells isolated from spleen and lymph nodes of unimmunized and EAE and EAE mice treated with 1,25(OH) 2 D 3. shown is a quantitative representation of blots by normalizing p-ERK protein level over ERK protein level. Data are representative of three independent experiments with at least five mice for each group. In A and C , data represent the mean ± S.D. ( error bars ) of three independent experiments each performed in triplicate. *, p

Techniques Used: Expressing, Mouse Assay, Isolation, Chromatin Immunoprecipitation

3) Product Images from "Tanshinone I enhances learning and memory, and ameliorates memory impairment in mice via the extracellular signal-regulated kinase signalling pathway"

Article Title: Tanshinone I enhances learning and memory, and ameliorates memory impairment in mice via the extracellular signal-regulated kinase signalling pathway

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2009.00378.x

The effects of tanshinone I on memory impairment induced by MK-801 via extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling. (A) Effect of tanshinone I (1, 2 or 4 mg·kg −1 , i.p.) on passive avoidance task latency. Tanshinone I was administered 40 min before, and MK-801 30 min before the acquisition trial. Data represent means ± SEM ( n = 10 per group). * P
Figure Legend Snippet: The effects of tanshinone I on memory impairment induced by MK-801 via extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling. (A) Effect of tanshinone I (1, 2 or 4 mg·kg −1 , i.p.) on passive avoidance task latency. Tanshinone I was administered 40 min before, and MK-801 30 min before the acquisition trial. Data represent means ± SEM ( n = 10 per group). * P

Techniques Used: Binding Assay

The effect of tanshinone I on memory impairment induced by diazepam via extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling. (A) Effect of tanshinone I (1, 2 and 4 mg·kg −1 , i.p.) on passive avoidance task latency. Tanshinone I was administered 40 min before, and diazepam 30 min before the acquisition trial. Data represent means ± SEM ( n = 10 per group). * P
Figure Legend Snippet: The effect of tanshinone I on memory impairment induced by diazepam via extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling. (A) Effect of tanshinone I (1, 2 and 4 mg·kg −1 , i.p.) on passive avoidance task latency. Tanshinone I was administered 40 min before, and diazepam 30 min before the acquisition trial. Data represent means ± SEM ( n = 10 per group). * P

Techniques Used: Binding Assay

The effects of tanshinone I on memory and extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling pathway involvement. Tanshinone I (0.5, 1, 2 or 4 mg·kg −1 , p.o.) increased passive avoidance task latency (A). Data represent means ± SEM ( n = 10 per group). * P
Figure Legend Snippet: The effects of tanshinone I on memory and extracellular signal-regulated kinase (ERK)–cAMP response element binding protein (CREB) signalling pathway involvement. Tanshinone I (0.5, 1, 2 or 4 mg·kg −1 , p.o.) increased passive avoidance task latency (A). Data represent means ± SEM ( n = 10 per group). * P

Techniques Used: Binding Assay

Photomicrographs of brain-derived neurotrophic factor (BDNF)-, phosphorylated cAMP response element binding protein (pCREB)- and phosphorylated extracellular signal-regulated kinase (pERK)-positive cells in the hippocampal CA1 region (A). Tanshinone I (4 mg·kg −1 , p.o.) was administered 40 min before transcardial perfusion. Summary data of the numbers of BDNF- (B), pCREB- (C) and pERK- (D) positive cells in CA1. Data represent means ± SEM for six determinations in each region for four animals. * P
Figure Legend Snippet: Photomicrographs of brain-derived neurotrophic factor (BDNF)-, phosphorylated cAMP response element binding protein (pCREB)- and phosphorylated extracellular signal-regulated kinase (pERK)-positive cells in the hippocampal CA1 region (A). Tanshinone I (4 mg·kg −1 , p.o.) was administered 40 min before transcardial perfusion. Summary data of the numbers of BDNF- (B), pCREB- (C) and pERK- (D) positive cells in CA1. Data represent means ± SEM for six determinations in each region for four animals. * P

Techniques Used: Derivative Assay, Binding Assay

Tanshinone I increases brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK) protein levels in the hippocampus. (A) pERK immunoreactivities in hippocampal tissues 40 min after treatment with tanshinone IIA (TanII, 4 mg·kg −1 , p.o.), cryptotanshinone (Cryp, 4 mg·kg −1 , p.o.), tanshinone I (TanI, 4 mg·kg −1 , p.o.) or 15,16-dihydrotanshinone I (Dihydro, 4 mg·kg −1 , p.o.). (B and C) BDNF, pCREB and pERK immunoreactivities as determined by Western blotting and densitometry. In graph C, the BDNF scale shows normalized ratios versus β-actin, whereas the other normalized ratios were calculated versus their respective total levels. Tanshinone I (1, 2 or 4 mg·kg −1 , p.o.) or vehicle solution (the same volume of 10% Tween 80) was administered to the mice 40 min before death. Data represent means ± SEM ( n = 4 per group). * P
Figure Legend Snippet: Tanshinone I increases brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK) protein levels in the hippocampus. (A) pERK immunoreactivities in hippocampal tissues 40 min after treatment with tanshinone IIA (TanII, 4 mg·kg −1 , p.o.), cryptotanshinone (Cryp, 4 mg·kg −1 , p.o.), tanshinone I (TanI, 4 mg·kg −1 , p.o.) or 15,16-dihydrotanshinone I (Dihydro, 4 mg·kg −1 , p.o.). (B and C) BDNF, pCREB and pERK immunoreactivities as determined by Western blotting and densitometry. In graph C, the BDNF scale shows normalized ratios versus β-actin, whereas the other normalized ratios were calculated versus their respective total levels. Tanshinone I (1, 2 or 4 mg·kg −1 , p.o.) or vehicle solution (the same volume of 10% Tween 80) was administered to the mice 40 min before death. Data represent means ± SEM ( n = 4 per group). * P

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

4) Product Images from "DNA methyltransferase 3a modulates chemosensitivity to gemcitabine and oxaliplatin via CHK1 and AKT in p53-deficient pancreatic cancer cells"

Article Title: DNA methyltransferase 3a modulates chemosensitivity to gemcitabine and oxaliplatin via CHK1 and AKT in p53-deficient pancreatic cancer cells

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.7923

Effect of DNMT3a downregulation by siRNA on activation of AKT, CHK1 and cell cycle arrest in Panc-1 cells. (A) Panc-1 cells were transfected with DNMT3a siRNA, then the cells were treated by GEM (5 µM) and OXA (5 µM) for 6 h. The expression level of p-AKT, AKT, p-ERK and ERK in negative control and DNMT3a knockdown cells was assessed by western blot analysis. (B) Following transfection with DNMT3a siRNA and treatment by GEM (5 µM) and OXA (5 µM) for 12 and 24 h, flow cytometry was applied to observe the cell cycle alteration. (C) Percentage of cells in S and G 0 /G 1 phases. (D) Western blot analysis of p-CHK1 and CHK1 expression in Panc-1 cells, which were transfected with DNMT3a siRNA, then treated with GEM (5 µM) and OXA (5 µM) for 6 h. Data are presented as the mean ± standard deviation. **P
Figure Legend Snippet: Effect of DNMT3a downregulation by siRNA on activation of AKT, CHK1 and cell cycle arrest in Panc-1 cells. (A) Panc-1 cells were transfected with DNMT3a siRNA, then the cells were treated by GEM (5 µM) and OXA (5 µM) for 6 h. The expression level of p-AKT, AKT, p-ERK and ERK in negative control and DNMT3a knockdown cells was assessed by western blot analysis. (B) Following transfection with DNMT3a siRNA and treatment by GEM (5 µM) and OXA (5 µM) for 12 and 24 h, flow cytometry was applied to observe the cell cycle alteration. (C) Percentage of cells in S and G 0 /G 1 phases. (D) Western blot analysis of p-CHK1 and CHK1 expression in Panc-1 cells, which were transfected with DNMT3a siRNA, then treated with GEM (5 µM) and OXA (5 µM) for 6 h. Data are presented as the mean ± standard deviation. **P

Techniques Used: Activation Assay, Transfection, Expressing, Negative Control, Western Blot, Flow Cytometry, Cytometry, Standard Deviation

5) Product Images from "Three-dimensional structure micelles of heparin-poloxamer improve the therapeutic effect of 17β-estradiol on endometrial regeneration for intrauterine adhesions in a rat model"

Article Title: Three-dimensional structure micelles of heparin-poloxamer improve the therapeutic effect of 17β-estradiol on endometrial regeneration for intrauterine adhesions in a rat model

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S137237

E 2 -HP hydrogel inhibits ER stress and activates the Akt and ERK1/2 pathways in the IUA rats. Notes: ( A ) The protein expressions of VEGF, GRP78, caspase-12, CHOP, p-Akt, and p-ERK in each group were tested with Western blotting. GAPDH was used as the loading control and for band density normalization. ( B ) The optical density analysis of GRP78, caspase-12, and CHOP protein. ** P
Figure Legend Snippet: E 2 -HP hydrogel inhibits ER stress and activates the Akt and ERK1/2 pathways in the IUA rats. Notes: ( A ) The protein expressions of VEGF, GRP78, caspase-12, CHOP, p-Akt, and p-ERK in each group were tested with Western blotting. GAPDH was used as the loading control and for band density normalization. ( B ) The optical density analysis of GRP78, caspase-12, and CHOP protein. ** P

Techniques Used: Western Blot

The activation of Akt and ERK1/2 is crucial for the protective effect of E 2 -HP hydrogel in H 2 O 2 -induced ER stress in EECs. Notes: ( A ) The protein expressions of GRP78, caspase-12, CHOP, p-Akt, and p-ERK1/2 in ER stress-induced apoptosis in EECs treated with E 2 -HP hydrogel and different inhibitors. GAPDH was used as the loading control and for band density normalization. ( B ) The optical density analysis of GRP78, caspase-12, and CHOP protein. ( C ) The optical density analysis of p-AKT and p-ERK protein. * P
Figure Legend Snippet: The activation of Akt and ERK1/2 is crucial for the protective effect of E 2 -HP hydrogel in H 2 O 2 -induced ER stress in EECs. Notes: ( A ) The protein expressions of GRP78, caspase-12, CHOP, p-Akt, and p-ERK1/2 in ER stress-induced apoptosis in EECs treated with E 2 -HP hydrogel and different inhibitors. GAPDH was used as the loading control and for band density normalization. ( B ) The optical density analysis of GRP78, caspase-12, and CHOP protein. ( C ) The optical density analysis of p-AKT and p-ERK protein. * P

Techniques Used: Activation Assay

6) Product Images from "Chebulinic acid inhibits smooth muscle cell migration by suppressing PDGF-Rβ phosphorylation and inhibiting matrix metalloproteinase-2 expression"

Article Title: Chebulinic acid inhibits smooth muscle cell migration by suppressing PDGF-Rβ phosphorylation and inhibiting matrix metalloproteinase-2 expression

Journal: Scientific Reports

doi: 10.1038/s41598-017-12221-w

Effect of CBA on PDGF-induced activation of downstream signalling pathways. HASMCs ( a and b ) and MOVAS-1 cells ( c and d ) were pre-treated with CBA for 30 min and then stimulated with PDGF-BB for 15 min. Cell lysates were separated by 10% SDS-PAGE and subjected to western blotting with the indicated antibodies. The membrane was stripped and reprobed with anti-tubulin, anti-Akt, and anti-ERK as loading controls. Relative protein levels were quantified by densitometric scanning and normalised to the corresponding total protein from three independent experiments; # P
Figure Legend Snippet: Effect of CBA on PDGF-induced activation of downstream signalling pathways. HASMCs ( a and b ) and MOVAS-1 cells ( c and d ) were pre-treated with CBA for 30 min and then stimulated with PDGF-BB for 15 min. Cell lysates were separated by 10% SDS-PAGE and subjected to western blotting with the indicated antibodies. The membrane was stripped and reprobed with anti-tubulin, anti-Akt, and anti-ERK as loading controls. Relative protein levels were quantified by densitometric scanning and normalised to the corresponding total protein from three independent experiments; # P

Techniques Used: Crocin Bleaching Assay, Activation Assay, SDS Page, Western Blot

7) Product Images from "TRAF3-interacting JNK-activating modulator promotes inflammation by stimulating translocation of Toll-like receptor 4 to lipid rafts"

Article Title: TRAF3-interacting JNK-activating modulator promotes inflammation by stimulating translocation of Toll-like receptor 4 to lipid rafts

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.003137

T3JAM positively regulates TLR4 signaling. A, Tnf α, Il6, and Ccl5 mRNA in PEMs from WT and T3jam −/− mice and treated with LPS (1 μg/ml) for periods of time ( horizontal axes ); results from three independent experiments were normalized to the expression of Gapdh in PBS-treated cells from WT mice. B, Tnf α, Il6, and Ccl5 mRNA in PEMs transduced with recombinant lentivirus for the overexpression of T3JAM or empty vector ( e.v. ) and treated with LPS (1 μg/ml) for periods of time ( horizontal axes ); results were normalized to those of PBS-treated cells transduced with empty vector ( e.v. ). C, immunoblot analysis of phosphorylated (p-) and total IKKβ, ERK, JNK, and p65, as well as IκBα, T3JAM, and β-actin; lysates of PEMs were from WT and T3jam −/− mice. Data are representative of three independent experiments with similar results. D, immunoblot analysis of phosphorylated (p-) and total IKKβ, ERK, JNK, and p65, as well as IκBα, T3JAM, and β-actin; lysates of PEMs were transfected with recombinant lentivirus for the overexpression of T3JAM or empty vector ( e.v. ), respectively. Data are representative of three independent experiments with similar results. E, survival of WT and T3jam −/− mice ( n = 10 per group) treated with LPS for periods of time ( horizontal axis ). p = 0.0014. F, Tnfα, Il-1β, and Il-6 levels in the lungs of WT and T3jam −/− mice ( n = 5 per group). Note: data are presented as means ± S.D. in A , B, and F. p values were calculated by unpaired Student's t test in A , B , and F and the log-rank test in E . *, p
Figure Legend Snippet: T3JAM positively regulates TLR4 signaling. A, Tnf α, Il6, and Ccl5 mRNA in PEMs from WT and T3jam −/− mice and treated with LPS (1 μg/ml) for periods of time ( horizontal axes ); results from three independent experiments were normalized to the expression of Gapdh in PBS-treated cells from WT mice. B, Tnf α, Il6, and Ccl5 mRNA in PEMs transduced with recombinant lentivirus for the overexpression of T3JAM or empty vector ( e.v. ) and treated with LPS (1 μg/ml) for periods of time ( horizontal axes ); results were normalized to those of PBS-treated cells transduced with empty vector ( e.v. ). C, immunoblot analysis of phosphorylated (p-) and total IKKβ, ERK, JNK, and p65, as well as IκBα, T3JAM, and β-actin; lysates of PEMs were from WT and T3jam −/− mice. Data are representative of three independent experiments with similar results. D, immunoblot analysis of phosphorylated (p-) and total IKKβ, ERK, JNK, and p65, as well as IκBα, T3JAM, and β-actin; lysates of PEMs were transfected with recombinant lentivirus for the overexpression of T3JAM or empty vector ( e.v. ), respectively. Data are representative of three independent experiments with similar results. E, survival of WT and T3jam −/− mice ( n = 10 per group) treated with LPS for periods of time ( horizontal axis ). p = 0.0014. F, Tnfα, Il-1β, and Il-6 levels in the lungs of WT and T3jam −/− mice ( n = 5 per group). Note: data are presented as means ± S.D. in A , B, and F. p values were calculated by unpaired Student's t test in A , B , and F and the log-rank test in E . *, p

Techniques Used: Mouse Assay, Expressing, Transduction, Recombinant, Over Expression, Plasmid Preparation, Transfection

8) Product Images from "Effects of hydrogen-rich saline on early acute kidney injury in severely burned rats by suppressing oxidative stress induced apoptosis and inflammation"

Article Title: Effects of hydrogen-rich saline on early acute kidney injury in severely burned rats by suppressing oxidative stress induced apoptosis and inflammation

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-015-0548-3

Effects of HS on signalling proteins post burn via western blotting analysis. HS relieved ROS-induced p38 phosphorylation and Akt inhibition ( a , b ). Both of p-JNK and p-ERK displayed marked elevations at the three time points, and HS administration decreased p-JNK and p-ERK expression ( c , d ). The sample size was n = 6 for each group. Following burn insult, NF-κB was markedly activated, and HS attenuated this activation ( e ). The results are expressed as the means ± SEMs. *p
Figure Legend Snippet: Effects of HS on signalling proteins post burn via western blotting analysis. HS relieved ROS-induced p38 phosphorylation and Akt inhibition ( a , b ). Both of p-JNK and p-ERK displayed marked elevations at the three time points, and HS administration decreased p-JNK and p-ERK expression ( c , d ). The sample size was n = 6 for each group. Following burn insult, NF-κB was markedly activated, and HS attenuated this activation ( e ). The results are expressed as the means ± SEMs. *p

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

9) Product Images from "RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma"

Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

Journal: Nature Communications

doi: 10.1038/ncomms15262

Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.
Figure Legend Snippet: Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.

Techniques Used: Mouse Assay, Western Blot, Expressing, Cell Culture, Activation Assay

RAF signalling is required for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a ) A melanoma from an untreated Braf f/f ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse was cut into small pieces and subcutaneously grafted into two groups of nude mice that were treated either with tamoxifen or vehicle for 2 weeks. The effect on tumour growth was assessed by measuring tumour volume over a 6-week period. Tumour volumes are plotted relative to the initial volume at the start of treatment. This experiment is representative of three independent experiments requiring 48 Swiss Nu/Nu females (6-week-old) for one primary tumour from a 1-year-old female on a SV129/C57Bl6 mixed genetic background. ( b ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels (pMEK and pERK, respectively) in protein lysates from culture in c on days 4 and 7 of 4OHT treatment compared to DMSO-treated culture. Total MEK, total ERK and β-actin are shown as a loading control. ( c ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ; Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour in response to 4OHT or DMSO for 9 days. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( d ) Cell cycle analysis by FACS from culture in c on day 6 of 4OHT treatment compared to DMSO-treated culture. Data are the mean value of three independent experiments. * P value
Figure Legend Snippet: RAF signalling is required for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a ) A melanoma from an untreated Braf f/f ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse was cut into small pieces and subcutaneously grafted into two groups of nude mice that were treated either with tamoxifen or vehicle for 2 weeks. The effect on tumour growth was assessed by measuring tumour volume over a 6-week period. Tumour volumes are plotted relative to the initial volume at the start of treatment. This experiment is representative of three independent experiments requiring 48 Swiss Nu/Nu females (6-week-old) for one primary tumour from a 1-year-old female on a SV129/C57Bl6 mixed genetic background. ( b ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels (pMEK and pERK, respectively) in protein lysates from culture in c on days 4 and 7 of 4OHT treatment compared to DMSO-treated culture. Total MEK, total ERK and β-actin are shown as a loading control. ( c ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ; Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour in response to 4OHT or DMSO for 9 days. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( d ) Cell cycle analysis by FACS from culture in c on day 6 of 4OHT treatment compared to DMSO-treated culture. Data are the mean value of three independent experiments. * P value

Techniques Used: Mouse Assay, Western Blot, Activation Assay, Cell Culture, Cell Cycle Assay, FACS

NRAS-mutated human melanoma cells require both BRAF and CRAF for ERK activation and proliferation. ( a ) Western blot analysis of BRAF and CRAF protein expression and pERK activation in NRAS-mutated human melanoma cell lines (WM1361, WM852 and Sbcl2) transfected with the scrambled control (−) or short interfering RNA to BRAF and/or CRAF (siBRAF1 and siCRAF1). Total ERK and β-actin are used as a loading control. ( b ) Proliferation rate in WM1361, WM852 and Sbcl2 cells transfected with scrambled control (scr), BRAF siRNA (B1), CRAF siRNA (C1) or BRAFsiRNA/CRAFsiRNA (B1C1) was measured after BrdU incorporation during 3 hours. ** P -value
Figure Legend Snippet: NRAS-mutated human melanoma cells require both BRAF and CRAF for ERK activation and proliferation. ( a ) Western blot analysis of BRAF and CRAF protein expression and pERK activation in NRAS-mutated human melanoma cell lines (WM1361, WM852 and Sbcl2) transfected with the scrambled control (−) or short interfering RNA to BRAF and/or CRAF (siBRAF1 and siCRAF1). Total ERK and β-actin are used as a loading control. ( b ) Proliferation rate in WM1361, WM852 and Sbcl2 cells transfected with scrambled control (scr), BRAF siRNA (B1), CRAF siRNA (C1) or BRAFsiRNA/CRAFsiRNA (B1C1) was measured after BrdU incorporation during 3 hours. ** P -value

Techniques Used: Activation Assay, Western Blot, Expressing, Transfection, Small Interfering RNA, BrdU Incorporation Assay

ARAF is required for the survival NRAS Q61K -induced murine melanoma cell lines in absence of BRAF and CRAF. ( a ) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( b ) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. ( c ) qRT–PCR analysis of ARAF expression in BRAF/CRAF Δ/Δ and parental control cultures. *** P value
Figure Legend Snippet: ARAF is required for the survival NRAS Q61K -induced murine melanoma cell lines in absence of BRAF and CRAF. ( a ) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( b ) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. ( c ) qRT–PCR analysis of ARAF expression in BRAF/CRAF Δ/Δ and parental control cultures. *** P value

Techniques Used: Double Knockout, Cell Culture, Western Blot, Expressing, Activation Assay, Quantitative RT-PCR

Vemurafenib induces ERK paradoxical activation in BRAF- and CRAF-decifient NRAS-induced melanoma by increasing ARAF kinase activity. ( a ) Western blot analysis of ERK activation (pERK) and ARAF, BRAF and CRAF protein expression in parental control and BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib (Vemu) or DMSO during 1 h. Total ERK and β-actin are used as loading controls. ( b ) ARAF in vitro kinase assays in BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib or DMSO during 1 h. ARAF was immunoprecipitated and its intrinsic kinase activity was measured on kinase-inactive MEK as substrate by western blotting using anti-pMEK antibody. Immune complexes and total cell extracts were immunoblotted with anti-ARAF, pMEK, MEK, pERK and ERK antibodies. β-actin was used as a loading control.
Figure Legend Snippet: Vemurafenib induces ERK paradoxical activation in BRAF- and CRAF-decifient NRAS-induced melanoma by increasing ARAF kinase activity. ( a ) Western blot analysis of ERK activation (pERK) and ARAF, BRAF and CRAF protein expression in parental control and BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib (Vemu) or DMSO during 1 h. Total ERK and β-actin are used as loading controls. ( b ) ARAF in vitro kinase assays in BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib or DMSO during 1 h. ARAF was immunoprecipitated and its intrinsic kinase activity was measured on kinase-inactive MEK as substrate by western blotting using anti-pMEK antibody. Immune complexes and total cell extracts were immunoblotted with anti-ARAF, pMEK, MEK, pERK and ERK antibodies. β-actin was used as a loading control.

Techniques Used: Activation Assay, Activity Assay, Western Blot, Expressing, In Vitro, Immunoprecipitation

10) Product Images from "Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor"

Article Title: Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor

Journal: Synapse (New York, N.Y.)

doi: 10.1002/syn.21549

Effect of cutamesine on protein levels of BDNF inside and outside cells One day after transfection with human proBDNF cDNA, B104 cells were treated with 1 μM of cutamesine overnight. Pro- and mature BDNF were immunoprecipitated in both cell lysate (IN) and culture medium (OUT), and detected by immunoblotting. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of control) are: control=100.0±7.1, cutamesine=70.6±8.8 (IN); control=100.0±6.2, cutamesine=174.7±21.4 (OUT).
Figure Legend Snippet: Effect of cutamesine on protein levels of BDNF inside and outside cells One day after transfection with human proBDNF cDNA, B104 cells were treated with 1 μM of cutamesine overnight. Pro- and mature BDNF were immunoprecipitated in both cell lysate (IN) and culture medium (OUT), and detected by immunoblotting. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of control) are: control=100.0±7.1, cutamesine=70.6±8.8 (IN); control=100.0±6.2, cutamesine=174.7±21.4 (OUT).

Techniques Used: Transfection, Immunoprecipitation

Effect of knockdown of Sig-1Rs on the secretion of BDNF from B104 cells A. Effect of Sig-1R knockdown on the BDNF protein levels inside and outside cells. B104 cells were transfected with proBDNF cDNA with or without Sig-1R siRNA and incubated for 2 days. CTR: control scramble siRNA. Pro- and mature BDNF inside (IN) and outside (OUT) cells were measured by immunoprecipitation as described in Fig. 5. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 4 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) are: CTR siRNA=100.0±4.2, Sig-1R siRNA=88.78±6.7 (IN); CTR siRNA=100.0±7.54, Sig-1R siRNA=65.8±12.7 (OUT). B. Pulse-chase experiment for monitoring synthesis and secretion of BDNF. B104 cells transfected with human BDNF cDNA for 1 day were pulse-labeled with 35 S-Met/Cys for 60 min followed by a chasing for 180 min. CTR: control scramble siRNA. BDNF were immunoprecipitated in both cell lysates (IN) and culture medium (OUT), and visualized by direct autoradiography. ERK and Sig-1Rs were measured in total cell lysates with immunoblotting. Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) 180 min after chasing are: CTR siRNA=100.0±0.1, Sig-1R siRNA=113.6±27.9 (IN); CTR siRNA=100.0±8.9, Sig-1R siRNA=53.4±8.0 (OUT).
Figure Legend Snippet: Effect of knockdown of Sig-1Rs on the secretion of BDNF from B104 cells A. Effect of Sig-1R knockdown on the BDNF protein levels inside and outside cells. B104 cells were transfected with proBDNF cDNA with or without Sig-1R siRNA and incubated for 2 days. CTR: control scramble siRNA. Pro- and mature BDNF inside (IN) and outside (OUT) cells were measured by immunoprecipitation as described in Fig. 5. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 4 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) are: CTR siRNA=100.0±4.2, Sig-1R siRNA=88.78±6.7 (IN); CTR siRNA=100.0±7.54, Sig-1R siRNA=65.8±12.7 (OUT). B. Pulse-chase experiment for monitoring synthesis and secretion of BDNF. B104 cells transfected with human BDNF cDNA for 1 day were pulse-labeled with 35 S-Met/Cys for 60 min followed by a chasing for 180 min. CTR: control scramble siRNA. BDNF were immunoprecipitated in both cell lysates (IN) and culture medium (OUT), and visualized by direct autoradiography. ERK and Sig-1Rs were measured in total cell lysates with immunoblotting. Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) 180 min after chasing are: CTR siRNA=100.0±0.1, Sig-1R siRNA=113.6±27.9 (IN); CTR siRNA=100.0±8.9, Sig-1R siRNA=53.4±8.0 (OUT).

Techniques Used: Transfection, Incubation, Immunoprecipitation, Pulse Chase, Labeling, Autoradiography

11) Product Images from "Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1"

Article Title: Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20042031

Receptor expression. (a) RT-PCR analysis of human peripheral blood leukocytes and glioma (DBTRG-05MG), monocytic (THP-1), lung epithelial (A549), hepatoma (HepG2), embryonic kidney (HEK293) cell lines, and brain and liver. (b) RT-PCR analysis of human peripheral blood monocytes exposed to either buffer alone, 10 ng/ml TNF-α, or 25 ng/ml IFN-γ for 6 h (gray bars) and 24 h (black bars). Expression levels were quantified by a National Institutes of Health image, normalized by GAPDH levels, and expressed as fold increase over vehicle-treated cells. (c) MAP kinase activation in human peripheral blood monocytic cells were exposed to RvE1 for 5 or 15 min and HEK-ChemR23 cells were treated with 100 nM RvE1 (E) or vehicle (V). (d) Pertussis toxin (PTX) blocks RvE1-induced ERK activation and NF-κB inhibition in HEK-ChemR23 cells. (e) EAR with HEK-ChemR23 cells exposed to vehicle (red line), 100 nM RvE1 (blue line), 10 μM chemerin peptide, or 10 μM ATP using Cytosensor microphysiometer. These responses were observed from three separate experiments. (f) Actions of RvE1 (red) and chemerin peptide (black) on [ 35 S]-GTP-γS binding to HEK293 cell membranes expressing human ChemR23. (g) Inhibition of TNF-α induced NF-κB luciferase activities by RvE1 (red) and chemerin peptide (black) on HEK293 cells expressing human ChemR23.
Figure Legend Snippet: Receptor expression. (a) RT-PCR analysis of human peripheral blood leukocytes and glioma (DBTRG-05MG), monocytic (THP-1), lung epithelial (A549), hepatoma (HepG2), embryonic kidney (HEK293) cell lines, and brain and liver. (b) RT-PCR analysis of human peripheral blood monocytes exposed to either buffer alone, 10 ng/ml TNF-α, or 25 ng/ml IFN-γ for 6 h (gray bars) and 24 h (black bars). Expression levels were quantified by a National Institutes of Health image, normalized by GAPDH levels, and expressed as fold increase over vehicle-treated cells. (c) MAP kinase activation in human peripheral blood monocytic cells were exposed to RvE1 for 5 or 15 min and HEK-ChemR23 cells were treated with 100 nM RvE1 (E) or vehicle (V). (d) Pertussis toxin (PTX) blocks RvE1-induced ERK activation and NF-κB inhibition in HEK-ChemR23 cells. (e) EAR with HEK-ChemR23 cells exposed to vehicle (red line), 100 nM RvE1 (blue line), 10 μM chemerin peptide, or 10 μM ATP using Cytosensor microphysiometer. These responses were observed from three separate experiments. (f) Actions of RvE1 (red) and chemerin peptide (black) on [ 35 S]-GTP-γS binding to HEK293 cell membranes expressing human ChemR23. (g) Inhibition of TNF-α induced NF-κB luciferase activities by RvE1 (red) and chemerin peptide (black) on HEK293 cells expressing human ChemR23.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Inhibition, Binding Assay, Luciferase

12) Product Images from "Pathological Comparisons of the Hippocampal Changes in the Transient and Permanent Middle Cerebral Artery Occlusion Rat Models"

Article Title: Pathological Comparisons of the Hippocampal Changes in the Transient and Permanent Middle Cerebral Artery Occlusion Rat Models

Journal: Frontiers in Neurology

doi: 10.3389/fneur.2019.01178

Ischemia induced cell stress and death related signaling changes (A) Representative Western blots of p-JNK, JNK, p-P38, P38, p-ERK, ERK. Densitometric analysis was expressed in relative to β-Actin ( n = 5/group). (B) Representative images of p-JNK immunoreactivity in CA1, CA2, and CA3 regions of hippocampus in I/R, p-MCAO and Dia p-MCAO group ( n = 5/group). scale bar = 100 μm. (C) Representative images of FJB histochemistry showing apoptotic cells; scale bar = 30 μm. Significant neuronal apoptosis in CA1, CA2 and CA3 region of hippocampus was observed in p-MCAO and Dia p-MCAO group ( n = 5/group). θ p
Figure Legend Snippet: Ischemia induced cell stress and death related signaling changes (A) Representative Western blots of p-JNK, JNK, p-P38, P38, p-ERK, ERK. Densitometric analysis was expressed in relative to β-Actin ( n = 5/group). (B) Representative images of p-JNK immunoreactivity in CA1, CA2, and CA3 regions of hippocampus in I/R, p-MCAO and Dia p-MCAO group ( n = 5/group). scale bar = 100 μm. (C) Representative images of FJB histochemistry showing apoptotic cells; scale bar = 30 μm. Significant neuronal apoptosis in CA1, CA2 and CA3 region of hippocampus was observed in p-MCAO and Dia p-MCAO group ( n = 5/group). θ p

Techniques Used: Western Blot

13) Product Images from "Functional interaction between p75NTR and TrkA: the endocytic trafficking of p75NTR is driven by TrkA and regulates TrkA-mediated signalling"

Article Title: Functional interaction between p75NTR and TrkA: the endocytic trafficking of p75NTR is driven by TrkA and regulates TrkA-mediated signalling

Journal: Biochemical Journal

doi: 10.1042/BJ20041155

Stimulation of p75 NTR by the apical addition of NGF induces TrkA phosphorylation and MAPK activation ( A ) E1A5 cells were stimulated by the apical (A) or basolateral (BL) addition of NGF. Inhibition of TrkA phosphorylation by 10 nM K252a was carried out 2 h before and during the stimulation time. Western blotting was carried out using an anti-(phosphorylated TrkA) (upper) or an anti-TrkA (lower) antibody. ( B ) E1A5 cells were grown and stimulated as described in ( A ). Western blotting was carried out using an anti-(phosphorylated ERK) (upper) or an anti-ERK (lower) antibody. ( C ) FRT cells expressing the p75 NTR mutant NTRPLAP were stimulated by apical loading of NGF for 15 min. Treated and control (CTRL) filters were subjected to indirect immunofluorescence using anti-p75 NTR monoclonal antibody and analysed by confocal microscopy. ( D ) FRT cells expressing NTRPLAP were stimulated from either the apical (A) or the basolateral (BL) surface with NGF. Western blotting was carried out using an anti-(phosphorylated TrkA) (upper) or an anti-TrkA (lower) antibody. These experiments were repeated more than three times, with similar results.
Figure Legend Snippet: Stimulation of p75 NTR by the apical addition of NGF induces TrkA phosphorylation and MAPK activation ( A ) E1A5 cells were stimulated by the apical (A) or basolateral (BL) addition of NGF. Inhibition of TrkA phosphorylation by 10 nM K252a was carried out 2 h before and during the stimulation time. Western blotting was carried out using an anti-(phosphorylated TrkA) (upper) or an anti-TrkA (lower) antibody. ( B ) E1A5 cells were grown and stimulated as described in ( A ). Western blotting was carried out using an anti-(phosphorylated ERK) (upper) or an anti-ERK (lower) antibody. ( C ) FRT cells expressing the p75 NTR mutant NTRPLAP were stimulated by apical loading of NGF for 15 min. Treated and control (CTRL) filters were subjected to indirect immunofluorescence using anti-p75 NTR monoclonal antibody and analysed by confocal microscopy. ( D ) FRT cells expressing NTRPLAP were stimulated from either the apical (A) or the basolateral (BL) surface with NGF. Western blotting was carried out using an anti-(phosphorylated TrkA) (upper) or an anti-TrkA (lower) antibody. These experiments were repeated more than three times, with similar results.

Techniques Used: Activation Assay, Inhibition, Western Blot, Expressing, Mutagenesis, Immunofluorescence, Confocal Microscopy

14) Product Images from "c-Src Signaling Induced by the Adapters Sin and Cas Is Mediated by Rap1 GTPase †"

Article Title: c-Src Signaling Induced by the Adapters Sin and Cas Is Mediated by Rap1 GTPase †

Journal: Molecular and Cellular Biology

doi:

Model for ligand-induced c-Src-dependent signal transduction leading to MAP kinase and transcriptional activation. Binding of the proline-rich motifs of Sin and Cas to the Src SH3 domain activates the c-Src tyrosine kinase activity, and the proteins become phosphorylated on tyrosine residues. Src-mediated phosphorylation of Sin and Cas results in the recruitment of the Crk-GEF signaling complex, which in turn activates Rap1 and ERK and induces SRE-mediated gene expression. On the other hand, mutations that disrupt the intramolecular interactions of Src, such as a point mutation on the C-terminal tyrosine 527, result in an open conformation of the enzyme, which then interacts nonspecifically with multiple cellular substrates. This, in turn, leads to the activation of at least two signaling cascades mediated by the Ras and Rap GTPases.
Figure Legend Snippet: Model for ligand-induced c-Src-dependent signal transduction leading to MAP kinase and transcriptional activation. Binding of the proline-rich motifs of Sin and Cas to the Src SH3 domain activates the c-Src tyrosine kinase activity, and the proteins become phosphorylated on tyrosine residues. Src-mediated phosphorylation of Sin and Cas results in the recruitment of the Crk-GEF signaling complex, which in turn activates Rap1 and ERK and induces SRE-mediated gene expression. On the other hand, mutations that disrupt the intramolecular interactions of Src, such as a point mutation on the C-terminal tyrosine 527, result in an open conformation of the enzyme, which then interacts nonspecifically with multiple cellular substrates. This, in turn, leads to the activation of at least two signaling cascades mediated by the Ras and Rap GTPases.

Techniques Used: Transduction, Activation Assay, Binding Assay, Activity Assay, Expressing, Mutagenesis

15) Product Images from "Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells"

Article Title: Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells

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

doi:

Effect of inhibitors of MAPK pathways on NECA-dependent IL-13 and VEGF secretion from BMMCs and HMC-1 cells. A , Western blot analysis of phosphorylation of ERK and p38 MAPK (phospho-ERK and phospho-p38, respectively) in BMMCs and HMC-1 cells that were either not stimulated (−) or stimulated with 10 μ M NECA for the indicated time period. Equal loading of proteins was verified by reprobing the same blot with total ERK and p38 MAPK Abs (ERK and p38, respectively). Blots are representative of three experiments. Effects of MEK inhibitor UO126 (●) and its inactive analog UO124 (○) on IL-13 ( B ) or VEGF ( D ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. Effects of p38 inhibitor SB202190 (●) and its inactive analog SB202474 (○) on IL-13 ( C ) or VEGF ( E ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. F , Effects of MEK inhibitor UO126 (10 μ M), p38 inhibitor SB202190 (10 μ M), and their inactive analogs UO124 (10 μ M) and SB202474 (10 μ M), respectively, on IL-13 secretion from BMMCs stimulated with 10 μ M NECA. Significant difference ( p
Figure Legend Snippet: Effect of inhibitors of MAPK pathways on NECA-dependent IL-13 and VEGF secretion from BMMCs and HMC-1 cells. A , Western blot analysis of phosphorylation of ERK and p38 MAPK (phospho-ERK and phospho-p38, respectively) in BMMCs and HMC-1 cells that were either not stimulated (−) or stimulated with 10 μ M NECA for the indicated time period. Equal loading of proteins was verified by reprobing the same blot with total ERK and p38 MAPK Abs (ERK and p38, respectively). Blots are representative of three experiments. Effects of MEK inhibitor UO126 (●) and its inactive analog UO124 (○) on IL-13 ( B ) or VEGF ( D ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. Effects of p38 inhibitor SB202190 (●) and its inactive analog SB202474 (○) on IL-13 ( C ) or VEGF ( E ) secretion from HMC-1 cells stimulated with 10 μ M NECA. Values are presented as mean percentage ± SEM of NECA-stimulated response from three experiments. F , Effects of MEK inhibitor UO126 (10 μ M), p38 inhibitor SB202190 (10 μ M), and their inactive analogs UO124 (10 μ M) and SB202474 (10 μ M), respectively, on IL-13 secretion from BMMCs stimulated with 10 μ M NECA. Significant difference ( p

Techniques Used: Western Blot

16) Product Images from "Hydrodynamic shear stress promotes epithelial-mesenchymal transition by downregulating ERK and GSK3β activities"

Article Title: Hydrodynamic shear stress promotes epithelial-mesenchymal transition by downregulating ERK and GSK3β activities

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-1071-2

Downregulation of the extracellular signal-related protein kinase (ERK) pathway is critical for the conversion of breast cancer cells derived from chemotherapy-treated patients (CT-PCs) into CT + shear stress (SS) displaying cancer stem-like cell (CSLC)/tumor-initiating cell (TIC) properties. a Transcriptional profile of the selected ERK-related genes ( Elk1 , Ets1 , Mcl1 , Tp53 , and Stat3 ). b Western blot analysis of p-ERK, ERK, p-p38, p38, p-JNK, JNK, and p53 from CT-PCs or CT + SS (10 days) n. c CT-PCs under +SS for 3 days were treated with PD98059 (mitogen-activated protein kinase (MEK) inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) for 24 h and subjected to sphere-formation assay (* p
Figure Legend Snippet: Downregulation of the extracellular signal-related protein kinase (ERK) pathway is critical for the conversion of breast cancer cells derived from chemotherapy-treated patients (CT-PCs) into CT + shear stress (SS) displaying cancer stem-like cell (CSLC)/tumor-initiating cell (TIC) properties. a Transcriptional profile of the selected ERK-related genes ( Elk1 , Ets1 , Mcl1 , Tp53 , and Stat3 ). b Western blot analysis of p-ERK, ERK, p-p38, p38, p-JNK, JNK, and p53 from CT-PCs or CT + SS (10 days) n. c CT-PCs under +SS for 3 days were treated with PD98059 (mitogen-activated protein kinase (MEK) inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) for 24 h and subjected to sphere-formation assay (* p

Techniques Used: Derivative Assay, Western Blot, Tube Formation Assay

17) Product Images from "Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells"

Article Title: Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.2009.10.1.23

The effects of a PI3K/Akt inhibitor and an MEK/ERK inhibitor on tetrandrine (TET)-treated A549 cells. The cells were treated with TET (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). Next, lysates were prepared and Western blot analysis was performed in order to determine protein expression levels.
Figure Legend Snippet: The effects of a PI3K/Akt inhibitor and an MEK/ERK inhibitor on tetrandrine (TET)-treated A549 cells. The cells were treated with TET (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). Next, lysates were prepared and Western blot analysis was performed in order to determine protein expression levels.

Techniques Used: Western Blot, Expressing

18) Product Images from "Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling"

Article Title: Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061523

IRAK-4 is critical for TLR-mediated activation of IRAK-1 and MAP kinases. (A) In vitro kinase assay for IRAK-1 activation. Peritoneal macrophages were stimulated with 10 ng/ml MALP-2 for the indicated periods. The cell lysates were prepared and immunoprecipitated with anti-IRAK-1 Ab, and the kinase activity of IRAK-1 was measured by in vitro kinase assay. The data shown are representative of three independent experiments. Auto, autophosphorylation. (B) Immunoblot analysis for the change in IRAK-1 expression in response to MALP-2 stimulation. Peritoneal macrophages from wild-type, IRAK-4 −/− , and IRAK-4 KN/KN mice were stimulated with MALP-2 for the indicated periods. Whole cell lysates were subject to immunoblot analysis using anti–IRAK-1. ERK1/2 levels are shown as loading control. (C–E) Macrophages from wild-type, IRAK-4 −/− , and IRAK-4 KN/KN mice were stimulated with MALP-2 for the indicated periods, and whole cell lysates were subject to immunoblot analysis using anti–phospho-JNK (C), anti–phospho-P38 (D), and anti–phospho-ERK (E). The blots of JNK, p38, and ERK are shown as loading controls. Similar results were obtained in three independent experiments.
Figure Legend Snippet: IRAK-4 is critical for TLR-mediated activation of IRAK-1 and MAP kinases. (A) In vitro kinase assay for IRAK-1 activation. Peritoneal macrophages were stimulated with 10 ng/ml MALP-2 for the indicated periods. The cell lysates were prepared and immunoprecipitated with anti-IRAK-1 Ab, and the kinase activity of IRAK-1 was measured by in vitro kinase assay. The data shown are representative of three independent experiments. Auto, autophosphorylation. (B) Immunoblot analysis for the change in IRAK-1 expression in response to MALP-2 stimulation. Peritoneal macrophages from wild-type, IRAK-4 −/− , and IRAK-4 KN/KN mice were stimulated with MALP-2 for the indicated periods. Whole cell lysates were subject to immunoblot analysis using anti–IRAK-1. ERK1/2 levels are shown as loading control. (C–E) Macrophages from wild-type, IRAK-4 −/− , and IRAK-4 KN/KN mice were stimulated with MALP-2 for the indicated periods, and whole cell lysates were subject to immunoblot analysis using anti–phospho-JNK (C), anti–phospho-P38 (D), and anti–phospho-ERK (E). The blots of JNK, p38, and ERK are shown as loading controls. Similar results were obtained in three independent experiments.

Techniques Used: Activation Assay, In Vitro, Kinase Assay, Immunoprecipitation, Activity Assay, Expressing, Mouse Assay

19) Product Images from "Physalis peruviana L. inhibits ovalbumin-induced airway inflammation by attenuating the activation of NF-κB and inflammatory molecules"

Article Title: Physalis peruviana L. inhibits ovalbumin-induced airway inflammation by attenuating the activation of NF-κB and inflammatory molecules

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4110

PP downregulates the activation of p38 mitogen-activated protein kinases in the lungs of mice with OVA-induced airway inflammation. (A) Levels of p38, ERK and JNK activation were determined by western blot analysis. Quantitative analysis of (B) p-p38, (C) p-JNK and (D) p-ERK was performed by densitometric analysis. # P
Figure Legend Snippet: PP downregulates the activation of p38 mitogen-activated protein kinases in the lungs of mice with OVA-induced airway inflammation. (A) Levels of p38, ERK and JNK activation were determined by western blot analysis. Quantitative analysis of (B) p-p38, (C) p-JNK and (D) p-ERK was performed by densitometric analysis. # P

Techniques Used: Activation Assay, Mouse Assay, Western Blot

20) Product Images from "A B56? mutation in lung cancer disrupts the p53-dependent tumor suppressor function of protein phosphatase 2A"

Article Title: A B56? mutation in lung cancer disrupts the p53-dependent tumor suppressor function of protein phosphatase 2A

Journal: Oncogene

doi: 10.1038/onc.2010.161

Mutant B56γ3 is unable to promote p53 Thr55 dephosphorylation. (A) Lysates of U2OS cells transfected with HA-tagged B56γ3, F395C, or Q392G/C398L (QC) mutant B56γ3, were either immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, PP2A A and C, HA, and vinculin (vinc) (left); or analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc) (right). Amino acid sequence of the p53-interaction domain of B56γ showing the cancer-derived F395C mutation is present on the top. (B) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, Q392G, C398L, Q392G/C398L (QC), or Q400S/Q401S (QQ) mutant B56γ3 were analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc). (C) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, or F395C mutant B56γ3 and either mock treated (M) or treated with ionizing radiation (IR) were analyzed for p53 Thr55 phosphorylation, HA, p53, and vinculin (vinc). (D) Lysates of U2OS cells transfected with varying amounts of HA-tagged F395C mutant B56γ3, were either mock (M) treated or treated with ionizing radiation (IR), then analyzed by western blot against endogenous (endo) and exogenous (exo) B56γ, as well as p53 and Thr55 phosphorylation. (E) Lysates of U2OS cells transfected with HA-tagged B56γ3 or F395C were immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, Cyclin G, ERK, Sgo, PP2A A, HA, and vinculin (vinc) antibodies.
Figure Legend Snippet: Mutant B56γ3 is unable to promote p53 Thr55 dephosphorylation. (A) Lysates of U2OS cells transfected with HA-tagged B56γ3, F395C, or Q392G/C398L (QC) mutant B56γ3, were either immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, PP2A A and C, HA, and vinculin (vinc) (left); or analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc) (right). Amino acid sequence of the p53-interaction domain of B56γ showing the cancer-derived F395C mutation is present on the top. (B) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, Q392G, C398L, Q392G/C398L (QC), or Q400S/Q401S (QQ) mutant B56γ3 were analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc). (C) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, or F395C mutant B56γ3 and either mock treated (M) or treated with ionizing radiation (IR) were analyzed for p53 Thr55 phosphorylation, HA, p53, and vinculin (vinc). (D) Lysates of U2OS cells transfected with varying amounts of HA-tagged F395C mutant B56γ3, were either mock (M) treated or treated with ionizing radiation (IR), then analyzed by western blot against endogenous (endo) and exogenous (exo) B56γ, as well as p53 and Thr55 phosphorylation. (E) Lysates of U2OS cells transfected with HA-tagged B56γ3 or F395C were immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, Cyclin G, ERK, Sgo, PP2A A, HA, and vinculin (vinc) antibodies.

Techniques Used: Mutagenesis, De-Phosphorylation Assay, Transfection, Immunoprecipitation, Western Blot, Sequencing, Derivative Assay, Plasmid Preparation

21) Product Images from "Chicken Embryos as a Potential New Model for Early Onset Type I Diabetes"

Article Title: Chicken Embryos as a Potential New Model for Early Onset Type I Diabetes

Journal: Journal of Diabetes Research

doi: 10.1155/2014/354094

The STZ-induced diabetic retina exhibits changed physiology. (a1-a3) ERGs were recorded from E20 embryos. (a1) Representative ERG waveforms from a control and a STZ-injected embryo. (a2) The amplitudes of a- and b-waves from STZ-injected embryos are significantly lower than the ones recorded from controls. (a3) The implicit time of both a- and b-waves from STZ-injected embryos are delayed, but there is a significant increase of the b-wave implicit time from STZ-injected embryos compared to the control. (b1-b2) Cone photoreceptors from control and STZ-injected embryonic retinas were dissociated and cultured at E18. The next day, patch-clamp recordings of L-VGCCs were carried out. (b1) The average data of voltage (mV) current density (pA/pF) relationship from the control and STZ-diabetic photoreceptors are shown. (b2) At both −40 mV and 0 mV, the L-VGCC current densities are significantly lower from STZ-diabetic photoreceptors compared to the control. (c) The STZ-injected diabetic retina has a significant decrease in the protein level of the L-VGCC α 1D subunit compared to the control. Total ERK serves as a loading control. * P
Figure Legend Snippet: The STZ-induced diabetic retina exhibits changed physiology. (a1-a3) ERGs were recorded from E20 embryos. (a1) Representative ERG waveforms from a control and a STZ-injected embryo. (a2) The amplitudes of a- and b-waves from STZ-injected embryos are significantly lower than the ones recorded from controls. (a3) The implicit time of both a- and b-waves from STZ-injected embryos are delayed, but there is a significant increase of the b-wave implicit time from STZ-injected embryos compared to the control. (b1-b2) Cone photoreceptors from control and STZ-injected embryonic retinas were dissociated and cultured at E18. The next day, patch-clamp recordings of L-VGCCs were carried out. (b1) The average data of voltage (mV) current density (pA/pF) relationship from the control and STZ-diabetic photoreceptors are shown. (b2) At both −40 mV and 0 mV, the L-VGCC current densities are significantly lower from STZ-diabetic photoreceptors compared to the control. (c) The STZ-injected diabetic retina has a significant decrease in the protein level of the L-VGCC α 1D subunit compared to the control. Total ERK serves as a loading control. * P

Techniques Used: Injection, Cell Culture, Patch Clamp

22) Product Images from "A New Functional Role for Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) in the Circadian Regulation of L-Type Voltage-Gated Calcium Channels in Avian Cone Photoreceptors"

Article Title: A New Functional Role for Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) in the Circadian Regulation of L-Type Voltage-Gated Calcium Channels in Avian Cone Photoreceptors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073315

The mTORC1 pathway is downstream of PI3K-AKT. Chick embryos (E11) were entrained in LD cycles for 7 days in ovo , and retinas were dissected, cultured, and kept in DD. On the second day of DD, cultured retinas were treated with 0.1% DMSO (control) or various inhibitors for 2 hr prior to harvest for immunoblotting at CT 4 and CT 16. (A) The phosphorylation of AKT at thr308 (pAKT thr308 ) was significantly higher in the control and rapamycin treated cells at CT 16 compared to other groups. “n.s.” indicates that there is no statical difference between the two CT 16 groups. * indicates statistical differences. (B) The phosphorylation of S6 (pS6), a downstream target of mTORC1, is significantly higher in the control at CT 16 (*) compared to all other groups. The level of pS6 in the control at CT 4 (#) is significantly higher than groups treated with rapamycin or PP242. (C) The levels of pAKT thr308 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. The levels of pAKT thr308 are significantly lower in both groups (CT 4 and CT 16) treated with LY294002 (#). (D) The levels of pS6 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. (E) The level of di-phosphorylated ERK (pERK) is significantly higher in the control at CT 16 (*), and there is no statistical difference between the control at CT 16 and LY294002 treated group at CT 16. The pERK levels in both PD98059 treated groups (CT 4 and CT 16) are significantly lower (#) compared to all other groups. (F) Treatment with rapamycin or PP242 does not affect the circadian rhythm of pERK, in which the pERK level is significantly higher at night (CT 16, *) compared to the day time level (CT 4). *, # p
Figure Legend Snippet: The mTORC1 pathway is downstream of PI3K-AKT. Chick embryos (E11) were entrained in LD cycles for 7 days in ovo , and retinas were dissected, cultured, and kept in DD. On the second day of DD, cultured retinas were treated with 0.1% DMSO (control) or various inhibitors for 2 hr prior to harvest for immunoblotting at CT 4 and CT 16. (A) The phosphorylation of AKT at thr308 (pAKT thr308 ) was significantly higher in the control and rapamycin treated cells at CT 16 compared to other groups. “n.s.” indicates that there is no statical difference between the two CT 16 groups. * indicates statistical differences. (B) The phosphorylation of S6 (pS6), a downstream target of mTORC1, is significantly higher in the control at CT 16 (*) compared to all other groups. The level of pS6 in the control at CT 4 (#) is significantly higher than groups treated with rapamycin or PP242. (C) The levels of pAKT thr308 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. The levels of pAKT thr308 are significantly lower in both groups (CT 4 and CT 16) treated with LY294002 (#). (D) The levels of pS6 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. (E) The level of di-phosphorylated ERK (pERK) is significantly higher in the control at CT 16 (*), and there is no statistical difference between the control at CT 16 and LY294002 treated group at CT 16. The pERK levels in both PD98059 treated groups (CT 4 and CT 16) are significantly lower (#) compared to all other groups. (F) Treatment with rapamycin or PP242 does not affect the circadian rhythm of pERK, in which the pERK level is significantly higher at night (CT 16, *) compared to the day time level (CT 4). *, # p

Techniques Used: In Ovo, Cell Culture

23) Product Images from "Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway"

Article Title: Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072079

Lentivirus-based MAPK/ERK pathway activation abolished the effect of OA on EMT of glioma cells. ( A ) 10 MOI of Lv-MEK or Lv-GFP was added to the cultures of U-87 MG and GT-1 # as well as 25 µg/mL (50 µM) of OA. 48 h later, total RNA was extracted from the cells and expression level of MEK mRNA was determined by qPCR. The experiments were performed for three times. GAPDH was selected as endogenous control. Their relative expression levels in OA-treated cells to untreated ones were shown as mean ± SD. ( B ) Under the same conditions, MAPK/ERK-responsive luciferase plasmids were transfected into U-87 MG and GT-1 # cells and Firefly luciferase activity was normalized by that of Renilla luciferase. The luciferase activity in untreated cells was selected as standards. These experiments were repeated for three times and the values were shown as mean ± SD. ( C ) Immunoblotting assay was perfermed to detect MEK and ERK proteins as well as their phosphorylated forms were also determined in 10 MOI of Lv-MEK or Lv-GFP-infected U-87 MG and GT-1 # cells under 25 µg/mL (50 µM) of OA treatment and GAPDH was selected as endogenous control. ( D ) 10 MOI of Lv-MEK or Lv-GFP was used to infect U-87 MG cells with or without 25 µg/mL (50 µM) of OA. 48 h later, transcripts of E-cadherin, N-cadherin, Vimentin and Twist1 were quantified. The experiments were performed for three times. GAPDH was selected as endogenous control. Their relative expression levels in the tested cells to ones treated by 25 µg/mL (50 µM) of OA and 10 MOI of Lv-GFP were shown as log 2 (mean ± SD). ( E ) E-cadherin, N-cadherin, Vimentin and Twist1 proteins were also determined in the cells treated simultaneously by lentivirus and 25 µg/mL (50 µM) of OA and GAPDH was selected as endogenous control.
Figure Legend Snippet: Lentivirus-based MAPK/ERK pathway activation abolished the effect of OA on EMT of glioma cells. ( A ) 10 MOI of Lv-MEK or Lv-GFP was added to the cultures of U-87 MG and GT-1 # as well as 25 µg/mL (50 µM) of OA. 48 h later, total RNA was extracted from the cells and expression level of MEK mRNA was determined by qPCR. The experiments were performed for three times. GAPDH was selected as endogenous control. Their relative expression levels in OA-treated cells to untreated ones were shown as mean ± SD. ( B ) Under the same conditions, MAPK/ERK-responsive luciferase plasmids were transfected into U-87 MG and GT-1 # cells and Firefly luciferase activity was normalized by that of Renilla luciferase. The luciferase activity in untreated cells was selected as standards. These experiments were repeated for three times and the values were shown as mean ± SD. ( C ) Immunoblotting assay was perfermed to detect MEK and ERK proteins as well as their phosphorylated forms were also determined in 10 MOI of Lv-MEK or Lv-GFP-infected U-87 MG and GT-1 # cells under 25 µg/mL (50 µM) of OA treatment and GAPDH was selected as endogenous control. ( D ) 10 MOI of Lv-MEK or Lv-GFP was used to infect U-87 MG cells with or without 25 µg/mL (50 µM) of OA. 48 h later, transcripts of E-cadherin, N-cadherin, Vimentin and Twist1 were quantified. The experiments were performed for three times. GAPDH was selected as endogenous control. Their relative expression levels in the tested cells to ones treated by 25 µg/mL (50 µM) of OA and 10 MOI of Lv-GFP were shown as log 2 (mean ± SD). ( E ) E-cadherin, N-cadherin, Vimentin and Twist1 proteins were also determined in the cells treated simultaneously by lentivirus and 25 µg/mL (50 µM) of OA and GAPDH was selected as endogenous control.

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Activity Assay, Infection

24) Product Images from "c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells"

Article Title: c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026851

c-Rel expression in nTreg precursors. c-Rel expression in nTreg precursors and nTregs. (A) Whole cell lysates from equivalent numbers of purified nTreg precursors and nTregs were analysed by Western blotting for c-Rel, Foxp3 and ERK (loading control) expression. The data is representative of three independent experiments. Purified nTreg precursors cultured in the absence or presence of IL-2 (B) or PMA plus ionomycin (C) for 2 hrs were subjected to sub-cellular fractionation and Western blots performed on nuclear and cytosolic fractions for c-Rel, ERK (cytosolic loading control) and histone H3 (nuclear loading control) expression. The data shown in panels B and C is representative of three independent experiments.
Figure Legend Snippet: c-Rel expression in nTreg precursors. c-Rel expression in nTreg precursors and nTregs. (A) Whole cell lysates from equivalent numbers of purified nTreg precursors and nTregs were analysed by Western blotting for c-Rel, Foxp3 and ERK (loading control) expression. The data is representative of three independent experiments. Purified nTreg precursors cultured in the absence or presence of IL-2 (B) or PMA plus ionomycin (C) for 2 hrs were subjected to sub-cellular fractionation and Western blots performed on nuclear and cytosolic fractions for c-Rel, ERK (cytosolic loading control) and histone H3 (nuclear loading control) expression. The data shown in panels B and C is representative of three independent experiments.

Techniques Used: Expressing, Purification, Western Blot, Cell Culture, Cell Fractionation

25) Product Images from "Role of IGF-1R in ameliorating apoptosis of GNE deficient cells"

Article Title: Role of IGF-1R in ameliorating apoptosis of GNE deficient cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-25510-9

Effect of GNE deficiency on IGF-1R downstream signaling pathway: Cells were grown in DCCM, followed by treatment with 5 nM IGF-1 for 10 min or treatment with 5 mM Sialic Acid or 0.5 μM PPP (IGF-1R inhibitor) for 24 h. ( A ) GNE mutant cells along with r-wt GNE and vector control cell lysates after treatment with or without 5 nM IGF-1 or 5 mM SA were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p-AKT/AKT, BAD/p-BAD, p-ERK/ERK1/2 and GAPDH antibodies. ( B ) GNE knockdown and vector control cell lysates with or without 5 nM IGF-1 treatment were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p- p-ERK/ERK1/2 and GAPDH antibodies. ( C ) GNE knockdown and vector control cell lysates after treatment with or without 5 nM IGF-1, 5 mM SA or 0.5 μM PPP (IGF-1R inhibitor) were separated on SDS–PAGE and immunoblotted with p-AKT/AKT, BCL2 and GAPDH antibodies.
Figure Legend Snippet: Effect of GNE deficiency on IGF-1R downstream signaling pathway: Cells were grown in DCCM, followed by treatment with 5 nM IGF-1 for 10 min or treatment with 5 mM Sialic Acid or 0.5 μM PPP (IGF-1R inhibitor) for 24 h. ( A ) GNE mutant cells along with r-wt GNE and vector control cell lysates after treatment with or without 5 nM IGF-1 or 5 mM SA were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p-AKT/AKT, BAD/p-BAD, p-ERK/ERK1/2 and GAPDH antibodies. ( B ) GNE knockdown and vector control cell lysates with or without 5 nM IGF-1 treatment were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p- p-ERK/ERK1/2 and GAPDH antibodies. ( C ) GNE knockdown and vector control cell lysates after treatment with or without 5 nM IGF-1, 5 mM SA or 0.5 μM PPP (IGF-1R inhibitor) were separated on SDS–PAGE and immunoblotted with p-AKT/AKT, BCL2 and GAPDH antibodies.

Techniques Used: Mutagenesis, Plasmid Preparation, SDS Page

Proposed Model: The figure shows that mutation in GNE inhibits sialic acid synthesis that may cause hyposialylation of IGF-1R. IGF-1R gets activated upon IGF-1 ligand binding and transduces the downstream signal to activate AKT that phosphorylate BAD leading to its dissociation from BCL2 which is anti-apoptotic signal to rescue cell cycle arrest and cell happily survives. Simultaneously, ERK may be downregulated in hyposialylated cells to cause dephosphorylation of BAD and its association with BCL2 continues to mediate mitochondrial dysfunction and cell apoptosis. A balance between cell survival and apoptosis pathway will determine the cell fate.
Figure Legend Snippet: Proposed Model: The figure shows that mutation in GNE inhibits sialic acid synthesis that may cause hyposialylation of IGF-1R. IGF-1R gets activated upon IGF-1 ligand binding and transduces the downstream signal to activate AKT that phosphorylate BAD leading to its dissociation from BCL2 which is anti-apoptotic signal to rescue cell cycle arrest and cell happily survives. Simultaneously, ERK may be downregulated in hyposialylated cells to cause dephosphorylation of BAD and its association with BCL2 continues to mediate mitochondrial dysfunction and cell apoptosis. A balance between cell survival and apoptosis pathway will determine the cell fate.

Techniques Used: Mutagenesis, Ligand Binding Assay, De-Phosphorylation Assay

26) Product Images from "Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice"

Article Title: Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice

Journal: Antioxidants

doi: 10.3390/antiox8100450

Alteration of the tropomyosin receptor kinase B (TrkB) receptor signaling pathway. Total tissue homogenates were prepared from the brain of Vehicle or GEGR treated SP-injected as described in Materials and Methods. Total protein (50 μg per sample) was immunoblotted with TrkB, p-TrkB, PI3K, p-PI3K, ERK, p-ERK, or β-actin antibodies. The intensity of each band was determined by using an imaging densitometer, and the relative levels of the proteins were based on the intensity of actin. Three samples were assayed in duplicate by western blotting. Data are reported as mean ± SD values. * p
Figure Legend Snippet: Alteration of the tropomyosin receptor kinase B (TrkB) receptor signaling pathway. Total tissue homogenates were prepared from the brain of Vehicle or GEGR treated SP-injected as described in Materials and Methods. Total protein (50 μg per sample) was immunoblotted with TrkB, p-TrkB, PI3K, p-PI3K, ERK, p-ERK, or β-actin antibodies. The intensity of each band was determined by using an imaging densitometer, and the relative levels of the proteins were based on the intensity of actin. Three samples were assayed in duplicate by western blotting. Data are reported as mean ± SD values. * p

Techniques Used: Injection, Imaging, Western Blot

27) Product Images from "Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice"

Article Title: Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice

Journal: The EMBO Journal

doi: 10.15252/embj.201695528

GM‐CSF receptor signaling is impaired in PIKfyve flox/flox lysm‐cre mice during AM development A AMs and IMs in lung, spleen, bone marrow, liver, and intraperitoneal cavity were isolated, and PIKfyve expression was measured by RT–PCR ( n = 3). B M‐CSF‐induced macrophages were cultured with GM‐CSF for 3 days, and CD11b + F4/80 + cells were stained with anti‐CD11c and anti‐Siglec‐F. Percentages of CD11c + Siglec‐F + cells in CD11b + F4/80 + cells are plotted as a bar graph ( n = 3). C M‐CSF‐induced macrophages were stimulated with GM‐CSF, and the time course of phosphorylation level of AKT, STAT5, NF‐κB p65, JNK, p38, and ERK was investigated by Western blot analysis using the indicated antibodies. D AMs or lungs were isolated, and the expression of Csf2 , Csfra, and Csfrb was measured by RT–PCR ( n = 3). E Expression of CD131 (GM‐CSF receptor β) in AM was measured by FACS ( n = 3). F, G Lungs were isolated at PND5. CD45 + cells were stained with anti‐CD11c and anti‐Siglec‐F, and CD45 + CD11c + Siglec‐F + cells were stained with anti‐pAKT (F) and anti‐pSTAT5 (G). Percentages of pAKT‐positive cells are plotted in a bar graph ( n = 4). H CD11c high Siglec‐F high cells were isolated from control, and CD11c high Siglec‐F + cells including Siglec‐F int cells were isolated from PIKfyve flox/flox lysm‐cre mice. Gene expression in AMs was measured by gene chip analysis, and the expression of transcription factors involved in macrophage development and function is shown. I M‐CSF‐induced macrophages were stimulated with GM‐CSF in the presence or absence of GSK690693, and the expression of Irf8 , Irf5, and Irf1 was measured by RT–PCR ( n = 4). Data information: Data are represented as mean ± SD. * P
Figure Legend Snippet: GM‐CSF receptor signaling is impaired in PIKfyve flox/flox lysm‐cre mice during AM development A AMs and IMs in lung, spleen, bone marrow, liver, and intraperitoneal cavity were isolated, and PIKfyve expression was measured by RT–PCR ( n = 3). B M‐CSF‐induced macrophages were cultured with GM‐CSF for 3 days, and CD11b + F4/80 + cells were stained with anti‐CD11c and anti‐Siglec‐F. Percentages of CD11c + Siglec‐F + cells in CD11b + F4/80 + cells are plotted as a bar graph ( n = 3). C M‐CSF‐induced macrophages were stimulated with GM‐CSF, and the time course of phosphorylation level of AKT, STAT5, NF‐κB p65, JNK, p38, and ERK was investigated by Western blot analysis using the indicated antibodies. D AMs or lungs were isolated, and the expression of Csf2 , Csfra, and Csfrb was measured by RT–PCR ( n = 3). E Expression of CD131 (GM‐CSF receptor β) in AM was measured by FACS ( n = 3). F, G Lungs were isolated at PND5. CD45 + cells were stained with anti‐CD11c and anti‐Siglec‐F, and CD45 + CD11c + Siglec‐F + cells were stained with anti‐pAKT (F) and anti‐pSTAT5 (G). Percentages of pAKT‐positive cells are plotted in a bar graph ( n = 4). H CD11c high Siglec‐F high cells were isolated from control, and CD11c high Siglec‐F + cells including Siglec‐F int cells were isolated from PIKfyve flox/flox lysm‐cre mice. Gene expression in AMs was measured by gene chip analysis, and the expression of transcription factors involved in macrophage development and function is shown. I M‐CSF‐induced macrophages were stimulated with GM‐CSF in the presence or absence of GSK690693, and the expression of Irf8 , Irf5, and Irf1 was measured by RT–PCR ( n = 4). Data information: Data are represented as mean ± SD. * P

Techniques Used: Mouse Assay, Affinity Magnetic Separation, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Staining, Western Blot, FACS, Chromatin Immunoprecipitation

28) Product Images from "Epstein-Barr Virus LMP1 Activates EGFR, STAT3, and ERK through Effects on PKC? ▿"

Article Title: Epstein-Barr Virus LMP1 Activates EGFR, STAT3, and ERK through Effects on PKC? ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01703-10

Model of LMP1 activation of EGFR, STAT3, and ERK. LMP1-CTAR1 induces intrinsic activation/phosphorylation of EGFR, which can be further activated by EGF to induce STAT3 tyrosine phosphorylation and the expression of STAT3 target genes (black arrows).
Figure Legend Snippet: Model of LMP1 activation of EGFR, STAT3, and ERK. LMP1-CTAR1 induces intrinsic activation/phosphorylation of EGFR, which can be further activated by EGF to induce STAT3 tyrosine phosphorylation and the expression of STAT3 target genes (black arrows).

Techniques Used: Activation Assay, Expressing

29) Product Images from "Ginsenoside Rg3 suppresses mast cell–mediated allergic inflammation via mitogen-activated protein kinase signaling pathway"

Article Title: Ginsenoside Rg3 suppresses mast cell–mediated allergic inflammation via mitogen-activated protein kinase signaling pathway

Journal: Journal of Ginseng Research

doi: 10.1016/j.jgr.2018.02.008

Ginsenoside Rg3 (G-Rg3) inhibited phosphorylation of MAPKs in activated mast cells. Phosphorylation of ERK, JNK, and p38 was detected by Western blotting. (A and B) HMC-1 cells (1 × 10 6 cells/well) were incubated with G-Rg3 and PDN (10 μM) for 30 min and stimulated with PMA (50 nM) + A23187 (1 μM) for 30 min. (C and D) DNP-IgE (100 ng/mL)–sensitized RBL-2H3 cells (1 × 10 6 cells/well) were incubated with G-Rg3 and PDN (10 μM) for 30 min and stimulated with DNP-BSA (10 μg/mL) for 30 min. Relative levels of MAPKs (B and D) were calculated using the Image J program (NIH, Bethesda, MD, USA). Results are the mean ± SD. # p
Figure Legend Snippet: Ginsenoside Rg3 (G-Rg3) inhibited phosphorylation of MAPKs in activated mast cells. Phosphorylation of ERK, JNK, and p38 was detected by Western blotting. (A and B) HMC-1 cells (1 × 10 6 cells/well) were incubated with G-Rg3 and PDN (10 μM) for 30 min and stimulated with PMA (50 nM) + A23187 (1 μM) for 30 min. (C and D) DNP-IgE (100 ng/mL)–sensitized RBL-2H3 cells (1 × 10 6 cells/well) were incubated with G-Rg3 and PDN (10 μM) for 30 min and stimulated with DNP-BSA (10 μg/mL) for 30 min. Relative levels of MAPKs (B and D) were calculated using the Image J program (NIH, Bethesda, MD, USA). Results are the mean ± SD. # p

Techniques Used: Western Blot, Incubation

30) Product Images from "The Roles of Versican V1 and V2 Isoforms in Cell Proliferation and Apoptosis"

Article Title: The Roles of Versican V1 and V2 Isoforms in Cell Proliferation and Apoptosis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-04-0295

Versican expression stimulates EGFR expression and Erk phosphorylation. (A) Equal amounts of cell lysate were separated on SDS-PAGE and subjected to Western blot analysis probed with anti-EGFR and antiactin (for loading control) antibodies. Cells transfected with V1 and V2mut expressed higher levels of EGFR compared with the other cell lines. (B) Total mRNA was prepared from the cells and subjected to RT-PCR using two primers (5′ cga ccc tca ggg acc gcg aga acc and 5′ gcc acc tcc tgg atg gtc ttt aag) generating a fragment (480 base pairs) of EGFR, followed by agarose gel electrophoresis. The V1- and V2mut-transfected cells produced a higher level of EGFR mRNA. (C) Phosphorylation of Erk (pErk) was analyzed by Western blotting using an antibody against phosphorylated Tyr204 of Erk. Consistent with the activation of EGFR, high levels of Erk phosphorylation were detected in V1- and V2mut-transfected cells. Low levels of Erk phosphorylation were observed in V2-transfected cells, and the phosphorylation level was not altered in V1mut-transfected cells. Erk protein levels were analyzed by Western blotting using Erk-specific antibody. Little difference was detected between the cell lines tested.
Figure Legend Snippet: Versican expression stimulates EGFR expression and Erk phosphorylation. (A) Equal amounts of cell lysate were separated on SDS-PAGE and subjected to Western blot analysis probed with anti-EGFR and antiactin (for loading control) antibodies. Cells transfected with V1 and V2mut expressed higher levels of EGFR compared with the other cell lines. (B) Total mRNA was prepared from the cells and subjected to RT-PCR using two primers (5′ cga ccc tca ggg acc gcg aga acc and 5′ gcc acc tcc tgg atg gtc ttt aag) generating a fragment (480 base pairs) of EGFR, followed by agarose gel electrophoresis. The V1- and V2mut-transfected cells produced a higher level of EGFR mRNA. (C) Phosphorylation of Erk (pErk) was analyzed by Western blotting using an antibody against phosphorylated Tyr204 of Erk. Consistent with the activation of EGFR, high levels of Erk phosphorylation were detected in V1- and V2mut-transfected cells. Low levels of Erk phosphorylation were observed in V2-transfected cells, and the phosphorylation level was not altered in V1mut-transfected cells. Erk protein levels were analyzed by Western blotting using Erk-specific antibody. Little difference was detected between the cell lines tested.

Techniques Used: Expressing, SDS Page, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Countercurrent Chromatography, Agarose Gel Electrophoresis, Produced, Activation Assay

31) Product Images from "Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis *"

Article Title: Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M804129200

Increased activation of ERK, Akt, and small GTPases contributes to the enhanced motility of SHP-2 D61G mutant cells. WT and SHP-2 D61G/D61G MEFs were preincubated with the MEK1 inhibitor PD98059 ( A ) or the PI3K inhibitor LY294002 ( B ) in serum-free
Figure Legend Snippet: Increased activation of ERK, Akt, and small GTPases contributes to the enhanced motility of SHP-2 D61G mutant cells. WT and SHP-2 D61G/D61G MEFs were preincubated with the MEK1 inhibitor PD98059 ( A ) or the PI3K inhibitor LY294002 ( B ) in serum-free

Techniques Used: Activation Assay, Mutagenesis

32) Product Images from "Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF"

Article Title: Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2016.280

Effect of miR-1246 on MEK-ERK signaling. A375P/Mdr cells were transiently transfected with a miR-1246 mimic or control RNA for 24 hours. Cell lysates were then prepared after treatment with the indicated concentrations of PLX4720 for 24 hours. The phosphorylated
Figure Legend Snippet: Effect of miR-1246 on MEK-ERK signaling. A375P/Mdr cells were transiently transfected with a miR-1246 mimic or control RNA for 24 hours. Cell lysates were then prepared after treatment with the indicated concentrations of PLX4720 for 24 hours. The phosphorylated

Techniques Used: Transfection

33) Product Images from "Discovery of novel non-ATP competitive FGFR1 inhibitors and evaluation of their anti-tumor activity in non-small cell lung cancer in vitro and in vivo"

Article Title: Discovery of novel non-ATP competitive FGFR1 inhibitors and evaluation of their anti-tumor activity in non-small cell lung cancer in vitro and in vivo

Journal: Oncotarget

doi:

NDGA analogs Aea4 and Aea25 inhibited FGFR1 activities (A) The profile of design and FGFR1 kinase inhibition assay of NDGA analogs. (B) Aea4 and Aea25 selectively inhibit FGFR1. Compounds were performed with caliper mobility shift assay for RTK inhibition, and the IC 50 values were calculated using conversion rates. The data were shown as a mean of 3-5 independent tests. (C and D) FGFR1 over-expressing 293 cells were pretreated with compounds at indicated concentrations or vehicle (0.1% DMSO), respectively. Then, cells were stimulated with bFGF (30 ng/mL) for 10 min, and the phosphorylation level of FGFR1 and ERK in cell lysates was measured by western blot analysis. The figures were representative of 3 separate experiments (C). The column figure shows the normalized optical density as a percentage of total protein control (D). Statistical significance relative to bFGF alone group was expressed, * P
Figure Legend Snippet: NDGA analogs Aea4 and Aea25 inhibited FGFR1 activities (A) The profile of design and FGFR1 kinase inhibition assay of NDGA analogs. (B) Aea4 and Aea25 selectively inhibit FGFR1. Compounds were performed with caliper mobility shift assay for RTK inhibition, and the IC 50 values were calculated using conversion rates. The data were shown as a mean of 3-5 independent tests. (C and D) FGFR1 over-expressing 293 cells were pretreated with compounds at indicated concentrations or vehicle (0.1% DMSO), respectively. Then, cells were stimulated with bFGF (30 ng/mL) for 10 min, and the phosphorylation level of FGFR1 and ERK in cell lysates was measured by western blot analysis. The figures were representative of 3 separate experiments (C). The column figure shows the normalized optical density as a percentage of total protein control (D). Statistical significance relative to bFGF alone group was expressed, * P

Techniques Used: Inhibition, Mobility Shift, Expressing, Western Blot

34) Product Images from "Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)"

Article Title: Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)

Journal: Journal of Oncology

doi: 10.1155/2009/627840

(a) Western blot of NF-kB : NF-kB expression in control (lane C) and ATRA treated (lane E) MCF-7 cells was observed by western blot analysis as before using anti-NF- κ B primary antibody. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells. (b), (c), and (d) Immunoblot assay of ERK, p-ERK, and VEGF: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (lane C) and presence of 30 μ M ATRA (lane E) for 24 hours in SFCM. Cells were collected and extracted in cell extraction buffer. ERK and PI-3K were immunoprecipitated from 150 μ g of protein from both control and ATRA-treated cell extract with anti-ERK protein G Agarose bead (Figures 7(b) and 7(c) ) and with anti-VEGF protein G Agarose bead ( Figure 7(d) ), keeping the samples for overnight at 4°C with shaking. In each case the resultant immune complex was washed thrice with PBS and the respective protein bound with antibody were eluted from the agarose bead using 1X sample buffer. Samples were then incubated in β -mercaptoethanol for 10 minutes at 90°C. Samples were subjected to electrophoresis on 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane by Western Blot. The membranes were incubated with anti-ERK ( Figure 7(b) ), anti-phospho ERK ( Figure 7(c) ), and anti-VEGF antibody ( Figure 7(d) ), respectively. The immunoblots were then incubated with alkaline phosphatase-coupled secondary antibodies and bands were visualized by NBT/BCIP substrate. Ig-G ( Figure 7(e) ) was used to confirm equal loading. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells.
Figure Legend Snippet: (a) Western blot of NF-kB : NF-kB expression in control (lane C) and ATRA treated (lane E) MCF-7 cells was observed by western blot analysis as before using anti-NF- κ B primary antibody. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells. (b), (c), and (d) Immunoblot assay of ERK, p-ERK, and VEGF: MCF-7 cells (300,000 cells/1.5 mL) were grown in absence (lane C) and presence of 30 μ M ATRA (lane E) for 24 hours in SFCM. Cells were collected and extracted in cell extraction buffer. ERK and PI-3K were immunoprecipitated from 150 μ g of protein from both control and ATRA-treated cell extract with anti-ERK protein G Agarose bead (Figures 7(b) and 7(c) ) and with anti-VEGF protein G Agarose bead ( Figure 7(d) ), keeping the samples for overnight at 4°C with shaking. In each case the resultant immune complex was washed thrice with PBS and the respective protein bound with antibody were eluted from the agarose bead using 1X sample buffer. Samples were then incubated in β -mercaptoethanol for 10 minutes at 90°C. Samples were subjected to electrophoresis on 7.5% SDS-PAGE. The proteins were transferred to nitrocellulose membrane by Western Blot. The membranes were incubated with anti-ERK ( Figure 7(b) ), anti-phospho ERK ( Figure 7(c) ), and anti-VEGF antibody ( Figure 7(d) ), respectively. The immunoblots were then incubated with alkaline phosphatase-coupled secondary antibodies and bands were visualized by NBT/BCIP substrate. Ig-G ( Figure 7(e) ) was used to confirm equal loading. Quantitative measurements of immunoblots were done by using Image J Launcher (version 1.4.3.67). (C) represents the expression of respective proteins in control cells whereas (E) represents expression in 30 μ M ATRA treated cells.

Techniques Used: Western Blot, Expressing, Immunoprecipitation, Incubation, Electrophoresis, SDS Page

35) Product Images from "Ghrelin Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells through the ERK Pathway"

Article Title: Ghrelin Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells through the ERK Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033126

ERK signaling pathway mediated the inhibitory effect of ghrelin on osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs). (A) The expression and silencing of growth hormone secretagog receptor (GHSR) on CVSMCs. Cells were incubated with scramble siRNA or GHSR siRNA for 72 h. Total cellular protein was subjected to western blot analysis using anti-GHSR antibody. The anti-GHSR antibody identified a band at 44 kDa. β-actin was used as the control. (B) The activation of extracellular signal-related kinase (ERK) under the silencing of GHSR. Cells were incubated with scramble siRNA or GHSR siRNA for 72 h prior to treatment with 10−6 mol/L ghrelin for 30 min. Total proteins were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown. (C) The activation of ERK under PD98059. Cells were incubated with PD98059 (10 µM) for 2 h prior to treatment with 10−6 mol/L of ghrelin for 30 min. Total proteins were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown. (D, E, F) The decreased alkaline phosphatase (ALP) activity, Runx2 mRNA, and calcium deposition mediated by the GHSR/ERK pathway. Cells were incubated with PD98059 (10 µM) for 2 h prior to treatment with 10−6 mol/L of ghrelin. Cells were also treated with the scramble siRNA or GHSR siRNA in the presence of 10−6 mol/L of ghrelin. ALP activity, Rnux2 mRNA, and calcium deposition were measured. The bars represent the mean ±standard deviation (SD) ( n = 3).
Figure Legend Snippet: ERK signaling pathway mediated the inhibitory effect of ghrelin on osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs). (A) The expression and silencing of growth hormone secretagog receptor (GHSR) on CVSMCs. Cells were incubated with scramble siRNA or GHSR siRNA for 72 h. Total cellular protein was subjected to western blot analysis using anti-GHSR antibody. The anti-GHSR antibody identified a band at 44 kDa. β-actin was used as the control. (B) The activation of extracellular signal-related kinase (ERK) under the silencing of GHSR. Cells were incubated with scramble siRNA or GHSR siRNA for 72 h prior to treatment with 10−6 mol/L ghrelin for 30 min. Total proteins were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown. (C) The activation of ERK under PD98059. Cells were incubated with PD98059 (10 µM) for 2 h prior to treatment with 10−6 mol/L of ghrelin for 30 min. Total proteins were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown. (D, E, F) The decreased alkaline phosphatase (ALP) activity, Runx2 mRNA, and calcium deposition mediated by the GHSR/ERK pathway. Cells were incubated with PD98059 (10 µM) for 2 h prior to treatment with 10−6 mol/L of ghrelin. Cells were also treated with the scramble siRNA or GHSR siRNA in the presence of 10−6 mol/L of ghrelin. ALP activity, Rnux2 mRNA, and calcium deposition were measured. The bars represent the mean ±standard deviation (SD) ( n = 3).

Techniques Used: Expressing, Incubation, Western Blot, Activation Assay, ALP Assay, Activity Assay, Standard Deviation

Effects of ghrelin on mitogen-activated protein kinase (MAPK) activation in calcifying vascular smooth muscle cells (CVSMCs). (A, B) Cells were exposed to 10−6 mol/L ghrelin for 0–60 min, or 50 µM hydrogen peroxide (H2O2) for 15 min as a positive control for p38 and JNK activation. Cell lysates were subjected to western blotting and incubated with antibodies against p-ERK, ERK, p-p38, p38, p-JNK, and JNK. The representative results are shown. (C) Cells exposed to 10−9 mol/L, 10 −8 mol/L, 10 −7 mol/L, and 10 −6 mol/L of ghrelin for 30 min. Cell lysates were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown.
Figure Legend Snippet: Effects of ghrelin on mitogen-activated protein kinase (MAPK) activation in calcifying vascular smooth muscle cells (CVSMCs). (A, B) Cells were exposed to 10−6 mol/L ghrelin for 0–60 min, or 50 µM hydrogen peroxide (H2O2) for 15 min as a positive control for p38 and JNK activation. Cell lysates were subjected to western blotting and incubated with antibodies against p-ERK, ERK, p-p38, p38, p-JNK, and JNK. The representative results are shown. (C) Cells exposed to 10−9 mol/L, 10 −8 mol/L, 10 −7 mol/L, and 10 −6 mol/L of ghrelin for 30 min. Cell lysates were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown.

Techniques Used: Activation Assay, Positive Control, Western Blot, Incubation

36) Product Images from "Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF"

Article Title: Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2016.280

Effect of miR-1246 on MEK-ERK signaling. A375P/Mdr cells were transiently transfected with a miR-1246 mimic or control RNA for 24 hours. Cell lysates were then prepared after treatment with the indicated concentrations of PLX4720 for 24 hours. The phosphorylated forms of MEK and ERK were detected by immunoblotting using anti–p-MEK and anti–p-ERK antibodies. The same blots were stripped and reprobed with anti-MEK and anti-ERK antibodies to confirm similar expression levels of MEK and ERK proteins in all lanes. The numbers listed below each band indicate the phosphorylated protein/total protein ratios determined using the Image Lab software. Data are representative of at least three independent experiments.
Figure Legend Snippet: Effect of miR-1246 on MEK-ERK signaling. A375P/Mdr cells were transiently transfected with a miR-1246 mimic or control RNA for 24 hours. Cell lysates were then prepared after treatment with the indicated concentrations of PLX4720 for 24 hours. The phosphorylated forms of MEK and ERK were detected by immunoblotting using anti–p-MEK and anti–p-ERK antibodies. The same blots were stripped and reprobed with anti-MEK and anti-ERK antibodies to confirm similar expression levels of MEK and ERK proteins in all lanes. The numbers listed below each band indicate the phosphorylated protein/total protein ratios determined using the Image Lab software. Data are representative of at least three independent experiments.

Techniques Used: Transfection, Expressing, Software

37) Product Images from "PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells"

Article Title: PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2019.8304

PYP1-4 restores the levels of Ras/Raf/ERK signaling pathway proteins in APAP-induced HepG2 cells. (A) HepG2 cells were incubated with 15 mM APAP with or without various concentrations of PYP1-4 for 18 h. The levels of Ras/Raf/ERK signaling pathway proteins (IGF-IR, SHC, GRB2, SOS, Ras, Raf, MEK and ERK) were determined by western blot analysis. (B) Bands were normalized to β-actin as an internal control, and the phosphorylated vs. total protein ratios were graphed. Data are presented as the mean ± SD of three independent experiments, and were subjected to two-way ANOVA. *P
Figure Legend Snippet: PYP1-4 restores the levels of Ras/Raf/ERK signaling pathway proteins in APAP-induced HepG2 cells. (A) HepG2 cells were incubated with 15 mM APAP with or without various concentrations of PYP1-4 for 18 h. The levels of Ras/Raf/ERK signaling pathway proteins (IGF-IR, SHC, GRB2, SOS, Ras, Raf, MEK and ERK) were determined by western blot analysis. (B) Bands were normalized to β-actin as an internal control, and the phosphorylated vs. total protein ratios were graphed. Data are presented as the mean ± SD of three independent experiments, and were subjected to two-way ANOVA. *P

Techniques Used: Incubation, Western Blot

38) Product Images from "Threonyl-tRNA Synthetase Promotes T Helper Type 1 Cell Responses by Inducing Dendritic Cell Maturation and IL-12 Production via an NF-κB Pathway"

Article Title: Threonyl-tRNA Synthetase Promotes T Helper Type 1 Cell Responses by Inducing Dendritic Cell Maturation and IL-12 Production via an NF-κB Pathway

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.571959

Threonyl-tRNA synthetase (TRS) activates dendritic cells (DCs) through the NF-κB and MAPK signaling pathways. (A) IκBα, IκBβ, and GAPDH expression levels, as measured by western blotting of DCs treated with TRS in a time-dependent manner (0–60 min). (B) The nuclear translocation of NF-κB p65, as detected by confocal laser scanning microscopy (LSM 800, Carl Zeiss, Oberkochen, Germany) of DCs treated with TRS (200 nM) for 1 h and then incubated with an anti-NF-κB p65 antibody (Ab), followed by staining with an Alexa Fluor 488-conjugated Ab and 4′,6-diamidino-2-phenylindole (DAPI). (C) DC culture method described in (A) . Western blotting for phosphorylated JNK, p38, and ERK or unphosphorylated JNK, p38, and ERK. (D) Levels of IκBα, IκBβ, NF-κB p65, and phosphorylated NF-κB p65, as detected via western blot analysis of DCs pretreated with the MAPK inhibitors (1, 10 μM) and treated for 30 min with TRS. (E) IL-12p40 levels in the supernatant, as detected via ELISA of DCs pretreated with the indicated inhibitors (0.1, 1, 10 μM) for 30 min and treated with TRS for 20 h. The media-treated DCs without inhibitors was analyzed as a control. Experiments were conducted three times independently and are represented as the mean ± SEM of results performed in triplicate (n = 3). Statistical significance was assessed using unpaired Student’s t -test; ### P
Figure Legend Snippet: Threonyl-tRNA synthetase (TRS) activates dendritic cells (DCs) through the NF-κB and MAPK signaling pathways. (A) IκBα, IκBβ, and GAPDH expression levels, as measured by western blotting of DCs treated with TRS in a time-dependent manner (0–60 min). (B) The nuclear translocation of NF-κB p65, as detected by confocal laser scanning microscopy (LSM 800, Carl Zeiss, Oberkochen, Germany) of DCs treated with TRS (200 nM) for 1 h and then incubated with an anti-NF-κB p65 antibody (Ab), followed by staining with an Alexa Fluor 488-conjugated Ab and 4′,6-diamidino-2-phenylindole (DAPI). (C) DC culture method described in (A) . Western blotting for phosphorylated JNK, p38, and ERK or unphosphorylated JNK, p38, and ERK. (D) Levels of IκBα, IκBβ, NF-κB p65, and phosphorylated NF-κB p65, as detected via western blot analysis of DCs pretreated with the MAPK inhibitors (1, 10 μM) and treated for 30 min with TRS. (E) IL-12p40 levels in the supernatant, as detected via ELISA of DCs pretreated with the indicated inhibitors (0.1, 1, 10 μM) for 30 min and treated with TRS for 20 h. The media-treated DCs without inhibitors was analyzed as a control. Experiments were conducted three times independently and are represented as the mean ± SEM of results performed in triplicate (n = 3). Statistical significance was assessed using unpaired Student’s t -test; ### P

Techniques Used: Expressing, Western Blot, Translocation Assay, Confocal Laser Scanning Microscopy, Incubation, Staining, Enzyme-linked Immunosorbent Assay

39) Product Images from "Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels"

Article Title: Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21176027

The effect of tepotinib on c-MET, β-catenin, ERK, and c-MYC protein levels in GC cells. The protein levels of phosphorylated and total c-MET, β-catenin, phosphorylated and total ERK, and c-MYC in MKN45, SNU620, MKN28, KATO III, and AGS cells were determined by Western blot analyses after treatment with tepotinib (10 nM) for 48 h. Data represent the means ± SD. Significance was evaluated by one-way ANOVA; * P
Figure Legend Snippet: The effect of tepotinib on c-MET, β-catenin, ERK, and c-MYC protein levels in GC cells. The protein levels of phosphorylated and total c-MET, β-catenin, phosphorylated and total ERK, and c-MYC in MKN45, SNU620, MKN28, KATO III, and AGS cells were determined by Western blot analyses after treatment with tepotinib (10 nM) for 48 h. Data represent the means ± SD. Significance was evaluated by one-way ANOVA; * P

Techniques Used: Western Blot

40) Product Images from "Role of IGF-1R in ameliorating apoptosis of GNE deficient cells"

Article Title: Role of IGF-1R in ameliorating apoptosis of GNE deficient cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-25510-9

Effect of GNE deficiency on IGF-1R downstream signaling pathway: Cells were grown in DCCM, followed by treatment with 5 nM IGF-1 for 10 min or treatment with 5 mM Sialic Acid or 0.5 μM PPP (IGF-1R inhibitor) for 24 h. ( A ) GNE mutant cells along with r-wt GNE and vector control cell lysates after treatment with or without 5 nM IGF-1 or 5 mM SA were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p-AKT/AKT, BAD/p-BAD, p-ERK/ERK1/2 and GAPDH antibodies. ( B ) GNE knockdown and vector control cell lysates with or without 5 nM IGF-1 treatment were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p- p-ERK/ERK1/2 and GAPDH antibodies. ( C ) GNE knockdown and vector control cell lysates after treatment with or without 5 nM IGF-1, 5 mM SA or 0.5 μM PPP (IGF-1R inhibitor) were separated on SDS–PAGE and immunoblotted with p-AKT/AKT, BCL2 and GAPDH antibodies.
Figure Legend Snippet: Effect of GNE deficiency on IGF-1R downstream signaling pathway: Cells were grown in DCCM, followed by treatment with 5 nM IGF-1 for 10 min or treatment with 5 mM Sialic Acid or 0.5 μM PPP (IGF-1R inhibitor) for 24 h. ( A ) GNE mutant cells along with r-wt GNE and vector control cell lysates after treatment with or without 5 nM IGF-1 or 5 mM SA were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p-AKT/AKT, BAD/p-BAD, p-ERK/ERK1/2 and GAPDH antibodies. ( B ) GNE knockdown and vector control cell lysates with or without 5 nM IGF-1 treatment were separated on SDS–PAGE and immunoblotted with p-IGF-1R/β-IGF-1R, p- p-ERK/ERK1/2 and GAPDH antibodies. ( C ) GNE knockdown and vector control cell lysates after treatment with or without 5 nM IGF-1, 5 mM SA or 0.5 μM PPP (IGF-1R inhibitor) were separated on SDS–PAGE and immunoblotted with p-AKT/AKT, BCL2 and GAPDH antibodies.

Techniques Used: Mutagenesis, Plasmid Preparation, SDS Page

Proposed Model: The figure shows that mutation in GNE inhibits sialic acid synthesis that may cause hyposialylation of IGF-1R. IGF-1R gets activated upon IGF-1 ligand binding and transduces the downstream signal to activate AKT that phosphorylate BAD leading to its dissociation from BCL2 which is anti-apoptotic signal to rescue cell cycle arrest and cell happily survives. Simultaneously, ERK may be downregulated in hyposialylated cells to cause dephosphorylation of BAD and its association with BCL2 continues to mediate mitochondrial dysfunction and cell apoptosis. A balance between cell survival and apoptosis pathway will determine the cell fate.
Figure Legend Snippet: Proposed Model: The figure shows that mutation in GNE inhibits sialic acid synthesis that may cause hyposialylation of IGF-1R. IGF-1R gets activated upon IGF-1 ligand binding and transduces the downstream signal to activate AKT that phosphorylate BAD leading to its dissociation from BCL2 which is anti-apoptotic signal to rescue cell cycle arrest and cell happily survives. Simultaneously, ERK may be downregulated in hyposialylated cells to cause dephosphorylation of BAD and its association with BCL2 continues to mediate mitochondrial dysfunction and cell apoptosis. A balance between cell survival and apoptosis pathway will determine the cell fate.

Techniques Used: Mutagenesis, Ligand Binding Assay, De-Phosphorylation Assay

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Western Blot:

Article Title: Depletion of cyclic‐GMP levels and inhibition of cGMP‐dependent protein kinase activate p21Cip1/p27Kip1 pathways and lead to renal fibrosis and dysfunction. Depletion of cyclic-GMP levels and inhibition of cGMP-dependent protein kinase activate p21Cip1/p27Kip1 pathways and lead to renal fibrosis and dysfunction
Article Snippet: .. In the Western blotting, primary antibodies were used as follows: cGK I (75 kDa; sc‐271766; 1:500; SCBT, Santa Cruz, CA, USA); cGK II (86 kDa; sc‐393126; 1:500); MKP‐1 (40 kDa; sc‐373841; 1:200; SCBT, Santa Cruz, CA, USA); Erk1/2 (44 kDa/42 kDa; sc‐514302; 1:250; SCBT, Santa Cruz, CA, USA); p38 (38 kDa; sc‐271120; 1:250; SCBT, Santa Cruz, CA, USA); p‐Erk1/2 (44 kDa/42 kDa; sc‐81492; 1:200; SCBT, Santa Cruz, CA, USA); p‐p38 (38 kDa; sc‐7973; 1:250; SCBT, Santa Cruz, CA, USA); p21Cip1 (21 kDa; sc‐6246; 1:250; SCBT, Santa Cruz, CA, USA); p27kip1 (27 kDa;sc‐1641; 1:200; SCBT, Santa Cruz, CA, USA); β‐actin (43 kDa; sc‐47778; 1:2000; SCBT, Santa Cruz, CA, USA); PCNA (36 kDa; sc‐56; 1:500; SCBT, Santa Cruz, CA, USA); HRP‐conjugated anti mouse IgG (sc‐516102; 1:1000; SCBT, Santa Cruz, CA, USA; G‐21040; 1:1000; Invitrogen, Eugene, OR, USA). .. 2.9 Assay of albumin and creatinine in urine samplesAlbumin levels were measured in 24‐hours urine samples collected from mice in a metabolic cage, using ELISA kit (Bethyl Laboratories, Montgomery, TX, USA).

Incubation:

Article Title: Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival, Migration and Proliferation
Article Snippet: .. The membranes were blocked with 5% albumin diluted in a Tris-buffered saline solution containing 1% Tween-20 (TBST) and then incubated overnight at 4°C in solutions with primary antibodies (1: 50) against Erk1/2 (sc-514302), phospho ERK1/2 (sc-81492), or PCNA (1: 10,000, sc-56) (Santa Cruz Biotechnology, Santa Cruz, CA, United States). .. The membranes were washed three times with TBST before an incubation for 1 h in solution with anti-mouse secondary antibody HRP-conjugated (1:10,000) (Santa Cruz Biotechnology, Santa Cruz, CA, United States).

other:

Article Title: Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy
Article Snippet: Reagents Mouse monoclonal antibody against cPLA2 (catalog n. sc-454) and mouse monoclonal p44/42 MAPK (Erk1/2, catalog sc-135900) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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    Santa Cruz Biotechnology rabbit anti erk ab
    Proposed mechanism of the combined action of FTS + MPA on EC cells Estrogens play an important role in the regulation of cell proliferation, differentiation, and function of the endometrium. They mediate their biological effects through two estrogen receptors in the nucleus, namely ERα and ERβ. ERs are transcription factors that are activated by phosphorylation and control gene expression. The activation is induced either in response to ligand binding or independently of ligand. Ras becomes active in the Ras-GTP form, which is upregulated by extracellular signals such as growth factors and hormones that bind the tyrosine kinase receptor. Once activated, Ras-GTP signals to multiple effector pathways that regulate proliferation, survival, metabolism, migration and shape of the cell. One of these pathways causes phosphorylation of <t>Akt,</t> which leads to cell survival, and another causes phosphorylation of <t>ERK,</t> leading to cell proliferation. Nuclear ERα is phosphorylated by pAkt at Ser167 and by pERK at Ser-118. Once phosphorylated, ERα is activated to target the transcription of genes (such as c-fos, ps2/TFF1 , and PR A+B ), leading to cell survival and proliferation. MPA increases ERα degradation, thereby reducing ERα in the cell (both in the nucleus and in the cytoplasm). FTS inhibits active Ras-GTP, leading to a decrease in pAkt and pERK, and hence a decrease in pERαSer118 and in pERαSer167, and finally a decrease in ERα gene transcription. These results showed that the combination of MPA + FTS inhibits proliferation of endometrial cells. ERα, estrogen receptor alpha; FTS, S-farnesylthiosalicylic acid; MPA, medroxyprogesterone acetate; PI3K, phosphatidylinositide 3-kinase; s118, serine-118; s167, serine-167.
    Rabbit Anti Erk Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The <t>4G10</t> (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho <t>ERK</t> and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.
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    Santa Cruz Biotechnology phospho extracellular signal regulated kinase
    The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, <t>phospho-c-Jun</t> N-terminal <t>kinase;</t> p-ERK, <t>phospho-extracellular</t> <t>signal-regulated</t> kinase.
    Phospho Extracellular Signal Regulated Kinase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proposed mechanism of the combined action of FTS + MPA on EC cells Estrogens play an important role in the regulation of cell proliferation, differentiation, and function of the endometrium. They mediate their biological effects through two estrogen receptors in the nucleus, namely ERα and ERβ. ERs are transcription factors that are activated by phosphorylation and control gene expression. The activation is induced either in response to ligand binding or independently of ligand. Ras becomes active in the Ras-GTP form, which is upregulated by extracellular signals such as growth factors and hormones that bind the tyrosine kinase receptor. Once activated, Ras-GTP signals to multiple effector pathways that regulate proliferation, survival, metabolism, migration and shape of the cell. One of these pathways causes phosphorylation of Akt, which leads to cell survival, and another causes phosphorylation of ERK, leading to cell proliferation. Nuclear ERα is phosphorylated by pAkt at Ser167 and by pERK at Ser-118. Once phosphorylated, ERα is activated to target the transcription of genes (such as c-fos, ps2/TFF1 , and PR A+B ), leading to cell survival and proliferation. MPA increases ERα degradation, thereby reducing ERα in the cell (both in the nucleus and in the cytoplasm). FTS inhibits active Ras-GTP, leading to a decrease in pAkt and pERK, and hence a decrease in pERαSer118 and in pERαSer167, and finally a decrease in ERα gene transcription. These results showed that the combination of MPA + FTS inhibits proliferation of endometrial cells. ERα, estrogen receptor alpha; FTS, S-farnesylthiosalicylic acid; MPA, medroxyprogesterone acetate; PI3K, phosphatidylinositide 3-kinase; s118, serine-118; s167, serine-167.

    Journal: Oncotarget

    Article Title: Growth of poorly differentiated endometrial carcinoma is inhibited by combined action of medroxyprogesterone acetate and the Ras inhibitor Salirasib

    doi:

    Figure Lengend Snippet: Proposed mechanism of the combined action of FTS + MPA on EC cells Estrogens play an important role in the regulation of cell proliferation, differentiation, and function of the endometrium. They mediate their biological effects through two estrogen receptors in the nucleus, namely ERα and ERβ. ERs are transcription factors that are activated by phosphorylation and control gene expression. The activation is induced either in response to ligand binding or independently of ligand. Ras becomes active in the Ras-GTP form, which is upregulated by extracellular signals such as growth factors and hormones that bind the tyrosine kinase receptor. Once activated, Ras-GTP signals to multiple effector pathways that regulate proliferation, survival, metabolism, migration and shape of the cell. One of these pathways causes phosphorylation of Akt, which leads to cell survival, and another causes phosphorylation of ERK, leading to cell proliferation. Nuclear ERα is phosphorylated by pAkt at Ser167 and by pERK at Ser-118. Once phosphorylated, ERα is activated to target the transcription of genes (such as c-fos, ps2/TFF1 , and PR A+B ), leading to cell survival and proliferation. MPA increases ERα degradation, thereby reducing ERα in the cell (both in the nucleus and in the cytoplasm). FTS inhibits active Ras-GTP, leading to a decrease in pAkt and pERK, and hence a decrease in pERαSer118 and in pERαSer167, and finally a decrease in ERα gene transcription. These results showed that the combination of MPA + FTS inhibits proliferation of endometrial cells. ERα, estrogen receptor alpha; FTS, S-farnesylthiosalicylic acid; MPA, medroxyprogesterone acetate; PI3K, phosphatidylinositide 3-kinase; s118, serine-118; s167, serine-167.

    Article Snippet: The lysates (75 μg protein) were immunoblotted with mouse anti-pan-Ras Ab (1:2,500, Calbiochem, San Diego, CA), rabbit anti-Akt Ab (1:1,000), rabbit anti-phospho-Akt (anti-pAkt) Ab (1:1,000), mouse anti-phospho-ERK (anti-pERK) Ab (1:10,000, Sigma-Aldrich), rabbit anti-ERK Ab (1:1,000) and, as a loading control, rabbit anti-β-tubulin Ab (1:500, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Activation Assay, Ligand Binding Assay, Migration

    FTS downregulates active Ras-GTP and its downstream signaling, leading to inhibition of cell proliferation in ECC1 and USPC1 EC cell lines ( a ) Dose-dependent decrease in the number of viable ECC1 or USPC1 cells as a function of FTS concentration. ECC1 and USPC1 cells were plated in 24-well plates, and treated after 24 hr with 0.1% DMSO (control) or FTS (100, 75, 50, or 25 μM). After 4 days the cells were counted. The IC 50 values of FTS in both cell lines were derived from the graph equations. ( b ) Immunoblots of Ras, Ras-GTP (active Ras), phospho-ERK, ERK, phospho-Akt, Akt, and β-tubulin (loading control) prepared from ECC1 and USPC1 control lysates and from lysates of ECC1 and USPC1 cells treated with 50μM FTS. ECC1 and USPC1 cells were plated in 10-cm plates and treated after 24 hr with 0.1% DMSO (control) or 50μM FTS. Three days later cells were lysed and subjected to western blotting with anti-pan-Ras, anti-Akt, anti-pAkt, anti-pERK, anti-ERK or anti-β-tubulin Abs (loading control). ( c ) FTS significantly decreases Ras-GTP, pERK, and pAkt both in ECC1 cells and ( d ) in USPC1 cells. There were no significant differences in total Ras, total ERK, total Akt or β-tubulin between control and FTS-treated cells. These results indicated that FTS acts in both cell lines as an inhibitor of active Ras and its downstream signaling. *, ** and *** are compared with the control for each cell line. * p

    Journal: Oncotarget

    Article Title: Growth of poorly differentiated endometrial carcinoma is inhibited by combined action of medroxyprogesterone acetate and the Ras inhibitor Salirasib

    doi:

    Figure Lengend Snippet: FTS downregulates active Ras-GTP and its downstream signaling, leading to inhibition of cell proliferation in ECC1 and USPC1 EC cell lines ( a ) Dose-dependent decrease in the number of viable ECC1 or USPC1 cells as a function of FTS concentration. ECC1 and USPC1 cells were plated in 24-well plates, and treated after 24 hr with 0.1% DMSO (control) or FTS (100, 75, 50, or 25 μM). After 4 days the cells were counted. The IC 50 values of FTS in both cell lines were derived from the graph equations. ( b ) Immunoblots of Ras, Ras-GTP (active Ras), phospho-ERK, ERK, phospho-Akt, Akt, and β-tubulin (loading control) prepared from ECC1 and USPC1 control lysates and from lysates of ECC1 and USPC1 cells treated with 50μM FTS. ECC1 and USPC1 cells were plated in 10-cm plates and treated after 24 hr with 0.1% DMSO (control) or 50μM FTS. Three days later cells were lysed and subjected to western blotting with anti-pan-Ras, anti-Akt, anti-pAkt, anti-pERK, anti-ERK or anti-β-tubulin Abs (loading control). ( c ) FTS significantly decreases Ras-GTP, pERK, and pAkt both in ECC1 cells and ( d ) in USPC1 cells. There were no significant differences in total Ras, total ERK, total Akt or β-tubulin between control and FTS-treated cells. These results indicated that FTS acts in both cell lines as an inhibitor of active Ras and its downstream signaling. *, ** and *** are compared with the control for each cell line. * p

    Article Snippet: The lysates (75 μg protein) were immunoblotted with mouse anti-pan-Ras Ab (1:2,500, Calbiochem, San Diego, CA), rabbit anti-Akt Ab (1:1,000), rabbit anti-phospho-Akt (anti-pAkt) Ab (1:1,000), mouse anti-phospho-ERK (anti-pERK) Ab (1:10,000, Sigma-Aldrich), rabbit anti-ERK Ab (1:1,000) and, as a loading control, rabbit anti-β-tubulin Ab (1:500, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Inhibition, Concentration Assay, Derivative Assay, Western Blot

    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Journal: Oncogene

    Article Title: Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML

    doi: 10.1038/onc.2016.41

    Figure Lengend Snippet: Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Article Snippet: Additional antibodies include: anti-phosphotyrosine 4G10 (Millipore, Darmstadt, Germany), anti-phospho AKT (Epitomics, Burlingame, CA, USA), anti-phospho ERK (Santa-Cruz Biotechnology Inc., Dallas, TX, USA), anti-phospho S6K and anti-S6K (Abcam, Cambridge, UK), anti-phospho p38 and anti-p38 (BD biosciences, Sparks, MD, USA) and anti-tubulin and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Concentration Assay, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

    p38 mediates the inhibition of anterograde FAT induced by SOD1ox ( a ) Immunoblotting analysis using activation-specific phosphoantibodies reveals a marked activation of p38 (p-p38) in axoplasms perfused with recombinant oxidized SOD1 (SOD1ox), compared to those perfused with recombinant unmodified WT-SOD1 (WT). In contrast, no changes were found in the activities of ERK (pERK) and GSK3 (pGSK3) in association with a specific SOD1 species. A monoclonal antibody against SOD1 (D3H5) 22 confirmed similar levels of SOD1 perfusion, and antibodies against kinesin-1 (KHC) provided a loading control for total levels of axoplasmic protein. Results from three independent experiments are shown (Squid 1-3). ( b ) Quantitation of results in (a) reveals an approximately 4-fold increase in the phosphorylation of p38 kinase (indicative of p38 activation) in SOD1ox-perfused axoplasms, compared to unmodified WT-SOD1-perfused axoplasms ( n =6, P

    Journal: Nature neuroscience

    Article Title: Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

    doi: 10.1038/nn.2660

    Figure Lengend Snippet: p38 mediates the inhibition of anterograde FAT induced by SOD1ox ( a ) Immunoblotting analysis using activation-specific phosphoantibodies reveals a marked activation of p38 (p-p38) in axoplasms perfused with recombinant oxidized SOD1 (SOD1ox), compared to those perfused with recombinant unmodified WT-SOD1 (WT). In contrast, no changes were found in the activities of ERK (pERK) and GSK3 (pGSK3) in association with a specific SOD1 species. A monoclonal antibody against SOD1 (D3H5) 22 confirmed similar levels of SOD1 perfusion, and antibodies against kinesin-1 (KHC) provided a loading control for total levels of axoplasmic protein. Results from three independent experiments are shown (Squid 1-3). ( b ) Quantitation of results in (a) reveals an approximately 4-fold increase in the phosphorylation of p38 kinase (indicative of p38 activation) in SOD1ox-perfused axoplasms, compared to unmodified WT-SOD1-perfused axoplasms ( n =6, P

    Article Snippet: Antibodies and reagents In our experiments, we used antibodies to SOD1 (PC077, the Binding Site; Calbiochem, 574597; SDG6 clone, Sigma), mutant-SOD1 (C4F6 and A9G3 ), KHC (H2) , phospho-p38 MAPK (Cell Signaling #9215), phospho-ERK (Santa Cruz #7383), and phospho-GSK3 (Santa Cruz #11757).

    Techniques: Inhibition, Activation Assay, Recombinant, Quantitation Assay

    The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, phospho-c-Jun N-terminal kinase; p-ERK, phospho-extracellular signal-regulated kinase.

    Journal: OncoTargets and therapy

    Article Title: Rhein inhibits malignant phenotypes of human renal cell carcinoma by impacting on MAPK/NF-κB signaling pathways

    doi: 10.2147/OTT.S153798

    Figure Lengend Snippet: The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, phospho-c-Jun N-terminal kinase; p-ERK, phospho-extracellular signal-regulated kinase.

    Article Snippet: Resultant blots were blocked with 5% skim milk and reacted with properly diluted primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz), phospho-extracellular signal-regulated kinase (Santa Cruz), total extracellular signal-regulated kinase (Santa Cruz), p-AKT (Cell Signaling Technology), phospho-c-Jun N-terminal kinase (Cell Signaling Technology), matrix metalloproteinase 9 (MMP9) (Cell Signaling Technology) and CCND1 (Cell Signaling Technology) for 1 h at room temperature.

    Techniques: Expressing, Software