Structured Review

Santa Cruz Biotechnology phosphorylated erk
Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on <t>ERK</t> activation. Relative density of <t>pERK/ERK</t> measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p
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1) Product Images from "Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines"

Article Title: Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines

Journal: Toxicology letters

doi: 10.1016/j.toxlet.2016.08.020

Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on ERK activation. Relative density of pERK/ERK measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p
Figure Legend Snippet: Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on ERK activation. Relative density of pERK/ERK measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p

Techniques Used: Activation Assay, Western Blot

2) Product Images from "Insulin-like Growth Factor-I Receptor Blockade Improves Outcome in Mouse Model of Lung Injury"

Article Title: Insulin-like Growth Factor-I Receptor Blockade Improves Outcome in Mouse Model of Lung Injury

Journal: American Journal of Respiratory and Critical Care Medicine

doi: 10.1164/rccm.200802-228OC

Mouse lung fibroblasts were serum starved overnight and then stimulated with insulin-like growth factor-I (100 ng/ml) for the indicated times. Cells were lysed, and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Each membrane was blotted with the indicated phospho-antibody [( A ) IGF-1R; ( B ) IRS-1, IRS-2 ( C ) AKT, ERK] and then stripped and reblotted with antibody to total protein.
Figure Legend Snippet: Mouse lung fibroblasts were serum starved overnight and then stimulated with insulin-like growth factor-I (100 ng/ml) for the indicated times. Cells were lysed, and proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Each membrane was blotted with the indicated phospho-antibody [( A ) IGF-1R; ( B ) IRS-1, IRS-2 ( C ) AKT, ERK] and then stripped and reblotted with antibody to total protein.

Techniques Used: Polyacrylamide Gel Electrophoresis

3) Product Images from "4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression"

Article Title: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2172-2

Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis
Figure Legend Snippet: Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

Techniques Used: Activation Assay, Concentration Assay, Western Blot

4) Product Images from "Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in erk-responsive lung cancer"

Article Title: Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in erk-responsive lung cancer

Journal: Oncotarget

doi:

Effects of the EGF-ERK signaling pathway on the regulation of miR-622 expression in relation to invasiveness of lung cancer A. A549-pLKO cells were treated with 5-aza-2′-deoxycytidine (5-AZA-dC; 10 or 20 μM) for 72 h. qRT-PCR was used to determine miR-622 level normalized to untreated cells. N.S., not significant. B. Western blotting for HIF-1α, ERK, phosphorylated ERK1/2 (p-ERK1/2), AKT, and phosphorylated AKT at T308 [p-AKT (Thr308)] in A549-pLKO/miR-622 stably expressing cells under serum starvation or restored by treatment with either 10% FBS or EGF (20 nM) for 6 h in a hypoxic state as described in Materials and methods. C. Quantitative results for the miR-622 level in A549-pLKO/miR-622 cells treated with EGF (20 nM) or 10% FBS was analyzed by qRT-PCR. RNU6B was used as an internal control. D. Boyden chamber assay for the assessment of invasion capacity of A549-pLKO/miR-622 cells treated with EGF (20 nM) or 10% FBS for 12 h. The results presented in panels (C) and (D) represent the mean ± S.D. of three independent experiments. * P
Figure Legend Snippet: Effects of the EGF-ERK signaling pathway on the regulation of miR-622 expression in relation to invasiveness of lung cancer A. A549-pLKO cells were treated with 5-aza-2′-deoxycytidine (5-AZA-dC; 10 or 20 μM) for 72 h. qRT-PCR was used to determine miR-622 level normalized to untreated cells. N.S., not significant. B. Western blotting for HIF-1α, ERK, phosphorylated ERK1/2 (p-ERK1/2), AKT, and phosphorylated AKT at T308 [p-AKT (Thr308)] in A549-pLKO/miR-622 stably expressing cells under serum starvation or restored by treatment with either 10% FBS or EGF (20 nM) for 6 h in a hypoxic state as described in Materials and methods. C. Quantitative results for the miR-622 level in A549-pLKO/miR-622 cells treated with EGF (20 nM) or 10% FBS was analyzed by qRT-PCR. RNU6B was used as an internal control. D. Boyden chamber assay for the assessment of invasion capacity of A549-pLKO/miR-622 cells treated with EGF (20 nM) or 10% FBS for 12 h. The results presented in panels (C) and (D) represent the mean ± S.D. of three independent experiments. * P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Stable Transfection, Boyden Chamber Assay

5) Product Images from "The Role of Zinc in the Modulation of Neuronal Proliferation and Apoptosis"

Article Title: The Role of Zinc in the Modulation of Neuronal Proliferation and Apoptosis

Journal: Neurotoxicity Research

doi: 10.1007/s12640-009-9067-4

Zn deficiency affects the distribution of events in the IMR-32 cell cycle. a Synchronized IMR-32 cells were incubated for 16 h in control non-chelated medium ( C ) or chelated media containing 1.5 or 15 μM Zn. The evaluation of cell cycle progression was done as described in “ Materials and methods ” section. The histograms show DNA staining by propidium iodide. The figure shows representative FACS profiles of the distribution of cells in G 0 –G 1 , S, and G 2 –M phases from four independent experiments. b Western blot for p53, cyclins E and D1, p-ERK, ERK, and tubulin in total cell fractions, and p21 (nuclear fractions), in synchronized cells incubated in control ( C ) or the media containing 1.5 μM Zn (1.5) for the indicated period of time. The figure shows representative images. Numbers under the figures are means from three independent experiments. Values for the 1.5 Zn group are significantly different ( P
Figure Legend Snippet: Zn deficiency affects the distribution of events in the IMR-32 cell cycle. a Synchronized IMR-32 cells were incubated for 16 h in control non-chelated medium ( C ) or chelated media containing 1.5 or 15 μM Zn. The evaluation of cell cycle progression was done as described in “ Materials and methods ” section. The histograms show DNA staining by propidium iodide. The figure shows representative FACS profiles of the distribution of cells in G 0 –G 1 , S, and G 2 –M phases from four independent experiments. b Western blot for p53, cyclins E and D1, p-ERK, ERK, and tubulin in total cell fractions, and p21 (nuclear fractions), in synchronized cells incubated in control ( C ) or the media containing 1.5 μM Zn (1.5) for the indicated period of time. The figure shows representative images. Numbers under the figures are means from three independent experiments. Values for the 1.5 Zn group are significantly different ( P

Techniques Used: Incubation, Staining, FACS, Western Blot

6) Product Images from "Isoflurane Protects Against Human Endothelial Cell Apoptosis by Inducing Sphingosine Kinase-1 via ERK MAPK"

Article Title: Isoflurane Protects Against Human Endothelial Cell Apoptosis by Inducing Sphingosine Kinase-1 via ERK MAPK

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms13010977

Inhibition of ERK MAPK phosphorylation with PD98059 (a selective MEK1 inhibitor) significantly reduced isoflurane-mediated induction of SK1 mRNA in EA.hy926 human endothelial cells. ( A ) Representative RT-PCR images for GAPDH, SK1 and SK2 from 4 independent experiments; ( B ) Densitometric quantifications of band intensities for SK1 and SK2 relative to GAPDH from RTPCR reactions. n = 4 per group. * p
Figure Legend Snippet: Inhibition of ERK MAPK phosphorylation with PD98059 (a selective MEK1 inhibitor) significantly reduced isoflurane-mediated induction of SK1 mRNA in EA.hy926 human endothelial cells. ( A ) Representative RT-PCR images for GAPDH, SK1 and SK2 from 4 independent experiments; ( B ) Densitometric quantifications of band intensities for SK1 and SK2 relative to GAPDH from RTPCR reactions. n = 4 per group. * p

Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction

Isoflurane increases ERK MAPK phosphorylation in EA.hy926 cells without changing total ERK expression. ( A ) Representative immunoblotting images of β-actin, phosphorylated ERK and total ERK from 4 independent experiments; ( B ) Densitometric quantifications of band intensities relative to beta-actin. n = 4 per group. * p
Figure Legend Snippet: Isoflurane increases ERK MAPK phosphorylation in EA.hy926 cells without changing total ERK expression. ( A ) Representative immunoblotting images of β-actin, phosphorylated ERK and total ERK from 4 independent experiments; ( B ) Densitometric quantifications of band intensities relative to beta-actin. n = 4 per group. * p

Techniques Used: Expressing

7) Product Images from "SiO2 Nanoparticles Induced Cytotoxicity by Oxidative Stress in Human Bronchial Epithelial Cell, Beas-2B"

Article Title: SiO2 Nanoparticles Induced Cytotoxicity by Oxidative Stress in Human Bronchial Epithelial Cell, Beas-2B

Journal: Environmental Health and Toxicology

doi: 10.5620/eht.2011.26.e2011013

Expression of intact and phosphorylated ERK, p38 and JNK in Beas-2B cells exposed to 1 mg/L SiO 2 nanoparticles. Densitometric values of expression of ERK, p38 and JNK were normalized using that of Actin and were presented as relative units compared to control. The control value was set to 1; data represent the mean ± standard error of the mean; n=3. ERK: extracellular signal-regulating kinase, JNK: c-Jun N-terminal kinase, SiO 2 : silicon dioxide. * p
Figure Legend Snippet: Expression of intact and phosphorylated ERK, p38 and JNK in Beas-2B cells exposed to 1 mg/L SiO 2 nanoparticles. Densitometric values of expression of ERK, p38 and JNK were normalized using that of Actin and were presented as relative units compared to control. The control value was set to 1; data represent the mean ± standard error of the mean; n=3. ERK: extracellular signal-regulating kinase, JNK: c-Jun N-terminal kinase, SiO 2 : silicon dioxide. * p

Techniques Used: Expressing

8) Product Images from "Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis"

Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

Journal: Technology in Cancer Research & Treatment

doi: 10.1177/1533034617754024

Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
Figure Legend Snippet: Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

Techniques Used:

9) Product Images from "Depression-Like Effect of Prenatal Buprenorphine Exposure in Rats"

Article Title: Depression-Like Effect of Prenatal Buprenorphine Exposure in Rats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082262

Buprenorphine decreased plasma BDNF and serotonin content and altered signaling molecule expression. Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21. Blood samples (n = 20 per group) were collected and subjected to ELISA for the measurement of BDNF (A) and serotonin (B). Brain cortical tissues were isolated. The obtained protein extracts (n = 5 per group) were subjected to Western blot analysis for the measurement of phosphorylated TrkB, TrkB, phosphorylated ERK, ERK, phosphorylated CREB, CREB, and β-tubulin (C). The obtained nuclear extracts (n = 5 per group) were subjected to EMSA for the measurement of CREB DNA binding activity (D). The protein extracts (n = 7 per group) were subjected to the enzymatic measurement of PKA activity (E). *p
Figure Legend Snippet: Buprenorphine decreased plasma BDNF and serotonin content and altered signaling molecule expression. Prenatal buprenorphine (0, 0.3, and 1 mg/kg/day) exposure was started from gestation day 7 and lasted for 14 days. After birth, the male and female pups were collected at postnatal day 21. Blood samples (n = 20 per group) were collected and subjected to ELISA for the measurement of BDNF (A) and serotonin (B). Brain cortical tissues were isolated. The obtained protein extracts (n = 5 per group) were subjected to Western blot analysis for the measurement of phosphorylated TrkB, TrkB, phosphorylated ERK, ERK, phosphorylated CREB, CREB, and β-tubulin (C). The obtained nuclear extracts (n = 5 per group) were subjected to EMSA for the measurement of CREB DNA binding activity (D). The protein extracts (n = 7 per group) were subjected to the enzymatic measurement of PKA activity (E). *p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Binding Assay, Activity Assay

10) Product Images from "Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells"

Article Title: Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells

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

doi: 10.12659/MSM.900956

Overexpression of CRT in SCs activated the PI3K/AKT and ERK/S6 signaling pathways. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. ( A ) expression levels of kinases in transfected cells. After transfection, total proteins extracted from transfected cells underwent Western blot analysis. ( B ) phosphorylation rates of different kinases in transfected cells. The band intensity was estimated by Image Lab™ software. The phosphorylation rate is expressed as the relative intensity of phosphorylated kinases/kinases and the final results were normalized by GAPDH. Data presented are the mean of 3 independent experiments. Error bars indicate SD. *** P
Figure Legend Snippet: Overexpression of CRT in SCs activated the PI3K/AKT and ERK/S6 signaling pathways. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. ( A ) expression levels of kinases in transfected cells. After transfection, total proteins extracted from transfected cells underwent Western blot analysis. ( B ) phosphorylation rates of different kinases in transfected cells. The band intensity was estimated by Image Lab™ software. The phosphorylation rate is expressed as the relative intensity of phosphorylated kinases/kinases and the final results were normalized by GAPDH. Data presented are the mean of 3 independent experiments. Error bars indicate SD. *** P

Techniques Used: Over Expression, Transfection, Negative Control, Expressing, Western Blot, Software

11) Product Images from "CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation"

Article Title: CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2012.136

CCN2 inactivates the Src-MAPK pathway without attenuation of the AKT-PI3K pathway. ( a ) ELISA results of CCN2 secretion in three lung cancer lines. ( b ) A time course of rCCN2 treatment in MAPK-phosphorylation (p-ERK-1/2) inhibition in above three lung adenocarcinoma cell lines and CL1-0/siCCN2 clones. ( c ) Western blotting analysis of AKT phosphorylation on S473 in CL1-5 cells treated with rCCN2 for indicated times. ( d ) In CCN2-stable transfectants as indicated, after MG132 treatment, whole-cell lysates were prepared for immunoprecipitation with anti-Src or anti-CCN2 followed by immunoblotting with anti-EGFR. α -tubulin was an internal control. ( e ) Western blotting assay in phospho-Src protein expression in CCN2 transfectants. ( f ) rCCN2-treated CL1-5 cells and phospho-Src protein expression was detected by western blotting assay. Total Src is loading control
Figure Legend Snippet: CCN2 inactivates the Src-MAPK pathway without attenuation of the AKT-PI3K pathway. ( a ) ELISA results of CCN2 secretion in three lung cancer lines. ( b ) A time course of rCCN2 treatment in MAPK-phosphorylation (p-ERK-1/2) inhibition in above three lung adenocarcinoma cell lines and CL1-0/siCCN2 clones. ( c ) Western blotting analysis of AKT phosphorylation on S473 in CL1-5 cells treated with rCCN2 for indicated times. ( d ) In CCN2-stable transfectants as indicated, after MG132 treatment, whole-cell lysates were prepared for immunoprecipitation with anti-Src or anti-CCN2 followed by immunoblotting with anti-EGFR. α -tubulin was an internal control. ( e ) Western blotting assay in phospho-Src protein expression in CCN2 transfectants. ( f ) rCCN2-treated CL1-5 cells and phospho-Src protein expression was detected by western blotting assay. Total Src is loading control

Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition, Clone Assay, Western Blot, Immunoprecipitation, Expressing

12) Product Images from "Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification"

Article Title: Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification

Journal: British Journal of Cancer

doi: 10.1038/bjc.2011.322

Phosphorylation of multiple RTKs in lung cancer cells with MET amplification. ( A ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence (control) or presence of PHA-665752 (500 n). Cell lysates were prepared and incubated with an RTK array for determination of the phosphorylation status of each enzyme. Each RTK is spotted in duplicate, and the pairs of dots in each corner of the array are positive controls. The numbered pairs of dots correspond to the indicated phosphorylated (p-) RTKs. ( B ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence or presence of PHA-665752 (PHA, 500 n), gefitinib (1 μ ), lapatinib (1 μ ), or vandetanib (1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total forms of MET, EGFR, HER2, HER3, RET, AKT, ERK, or STAT3 or with those to β -actin (loading control). ( C ) The indicated cell lines were deprived of serum for 24 h and then incubated for 2 h in the absence (Con) or presence of gefitinib (Gef, 1 μ ), lapatinib (Lap, 1 μ ), or vandetanib (Van, 1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins.
Figure Legend Snippet: Phosphorylation of multiple RTKs in lung cancer cells with MET amplification. ( A ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence (control) or presence of PHA-665752 (500 n). Cell lysates were prepared and incubated with an RTK array for determination of the phosphorylation status of each enzyme. Each RTK is spotted in duplicate, and the pairs of dots in each corner of the array are positive controls. The numbered pairs of dots correspond to the indicated phosphorylated (p-) RTKs. ( B ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence or presence of PHA-665752 (PHA, 500 n), gefitinib (1 μ ), lapatinib (1 μ ), or vandetanib (1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total forms of MET, EGFR, HER2, HER3, RET, AKT, ERK, or STAT3 or with those to β -actin (loading control). ( C ) The indicated cell lines were deprived of serum for 24 h and then incubated for 2 h in the absence (Con) or presence of gefitinib (Gef, 1 μ ), lapatinib (Lap, 1 μ ), or vandetanib (Van, 1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins.

Techniques Used: Amplification, Incubation

13) Product Images from "Differential regulation of interleukin-8 and human beta-defensin 2 in Pseudomonas aeruginosa-infected intestinal epithelial cells"

Article Title: Differential regulation of interleukin-8 and human beta-defensin 2 in Pseudomonas aeruginosa-infected intestinal epithelial cells

Journal: BMC Microbiology

doi: 10.1186/s12866-014-0275-6

The proteins expression of intracellular signaling pathways in P. aeruginosa -infected SW480 cells. SW480 cells were left uninfected (CON), or infected with wild-type P. aeruginosa strain PAO1 for the times indicated. Activation of the ERK, JNK, p38, Akt and IκB were analyzed in whole cell protein by immunoblotting with antibodies to phosphorylated (p) ERK, JNK, p38, Akt and IκB. The results shown are representative of three separate experiments. GAPDH worked as a normalization of cytosolic protein.
Figure Legend Snippet: The proteins expression of intracellular signaling pathways in P. aeruginosa -infected SW480 cells. SW480 cells were left uninfected (CON), or infected with wild-type P. aeruginosa strain PAO1 for the times indicated. Activation of the ERK, JNK, p38, Akt and IκB were analyzed in whole cell protein by immunoblotting with antibodies to phosphorylated (p) ERK, JNK, p38, Akt and IκB. The results shown are representative of three separate experiments. GAPDH worked as a normalization of cytosolic protein.

Techniques Used: Expressing, Infection, Activation Assay

The involvement of ERK and PI3K/Akt signal pathways in P. aeruginosa -induced IL-8 in SW480 cells. (A) Effect of ERK and PI3K inhibition on P. aeruginosa -induced IL-8 and hBD-2 secretion. SW480 cells were left untreated, or treated with 25 μM PD98059 (PD) and 50 μM LY294002 (LY) for one hour. They were then infected with the wild-type P. aeruginosa strain PAO1 for 7 hours. Supernatant was analyzed by ELISA for IL-8 and hBD-2. The amount of IL-8 and hBD-2 produced is shown as the fold increase over uninfected control (CON) cells. (B) Effect of ERK and PI3K inhibition on P. aeruginosa -induced IL-8 and hBD-2 mRNA. SW480 cells were left untreated, or treated with 25 μM PD98059 (PD) and 50 μM LY294002 (LY). They were then infected with the wild-type P. aeruginosa strain PAO1 for 7 hours. Total RNA was prepared and analyzed by real-time quantitative PCR to estimate amounts of IL-8 and hBD-2 transcript. The amount of IL-8 and hBD-2 mRNA produced, normalized to the corresponding amount of GAPDH transcript, is shown as the fold increase over uninfected control (CON) cells. Results are represented as means ± S.E.M. for at least three determinations from independent experiments. (* p
Figure Legend Snippet: The involvement of ERK and PI3K/Akt signal pathways in P. aeruginosa -induced IL-8 in SW480 cells. (A) Effect of ERK and PI3K inhibition on P. aeruginosa -induced IL-8 and hBD-2 secretion. SW480 cells were left untreated, or treated with 25 μM PD98059 (PD) and 50 μM LY294002 (LY) for one hour. They were then infected with the wild-type P. aeruginosa strain PAO1 for 7 hours. Supernatant was analyzed by ELISA for IL-8 and hBD-2. The amount of IL-8 and hBD-2 produced is shown as the fold increase over uninfected control (CON) cells. (B) Effect of ERK and PI3K inhibition on P. aeruginosa -induced IL-8 and hBD-2 mRNA. SW480 cells were left untreated, or treated with 25 μM PD98059 (PD) and 50 μM LY294002 (LY). They were then infected with the wild-type P. aeruginosa strain PAO1 for 7 hours. Total RNA was prepared and analyzed by real-time quantitative PCR to estimate amounts of IL-8 and hBD-2 transcript. The amount of IL-8 and hBD-2 mRNA produced, normalized to the corresponding amount of GAPDH transcript, is shown as the fold increase over uninfected control (CON) cells. Results are represented as means ± S.E.M. for at least three determinations from independent experiments. (* p

Techniques Used: Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Produced, Real-time Polymerase Chain Reaction

14) Product Images from "Chebulagic acid inhibits the LPS-induced expression of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation"

Article Title: Chebulagic acid inhibits the LPS-induced expression of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2015.2447

Effect of CA on the activation of mitogen-activated protein kinase signaling in LPS-stimulated EA.hy926 cells. (A) Western blot analysis of p38, JNK and ERK with or without phosphorylation in CA- and LPS-treated EA.hy926 cells. Percentage of phosphorylated
Figure Legend Snippet: Effect of CA on the activation of mitogen-activated protein kinase signaling in LPS-stimulated EA.hy926 cells. (A) Western blot analysis of p38, JNK and ERK with or without phosphorylation in CA- and LPS-treated EA.hy926 cells. Percentage of phosphorylated

Techniques Used: Activation Assay, Western Blot

15) Product Images from "Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner"

Article Title: Silibinin Inhibits Platelet-Derived Growth Factor-Driven Cell Proliferation via Downregulation of N-Glycosylation in Human Tenon's Fibroblasts in a Proteasome-Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0168765

Effects of silibinin on platelet-derived growth factor (PDGF)-regulated signaling pathways in Human Tenon's fibroblasts (HTFs). HTFs were pretreated with either vehicle or silibinin (50 or 100 μM) for 24 h. The cells were then treated with PDGF for 30 min. (A) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-extracellular-signal-regulated kinase (ERK) 1/2, total ERK 1/2, and GAPDH. Mean phosphorylated-ERK 1/2 levels were determined using densitometric analysis and normalized to total ERK 1/2. (B) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-signal transducer and activator of transcription 1 (STAT1), total STAT1, and GAPDH. Mean phosphorylated-STAT1 levels were determined using densitometric analysis and normalized to total STAT1. (C) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-STAT3, total STAT3, and GAPDH. Mean phosphorylated-STAT3 levels were determined using densitometric analysis and normalized to total STAT3. The data are presented as means ± SEM of three independent experiments. Asterisks (* and **) indicate responses that are significantly different (p
Figure Legend Snippet: Effects of silibinin on platelet-derived growth factor (PDGF)-regulated signaling pathways in Human Tenon's fibroblasts (HTFs). HTFs were pretreated with either vehicle or silibinin (50 or 100 μM) for 24 h. The cells were then treated with PDGF for 30 min. (A) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-extracellular-signal-regulated kinase (ERK) 1/2, total ERK 1/2, and GAPDH. Mean phosphorylated-ERK 1/2 levels were determined using densitometric analysis and normalized to total ERK 1/2. (B) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-signal transducer and activator of transcription 1 (STAT1), total STAT1, and GAPDH. Mean phosphorylated-STAT1 levels were determined using densitometric analysis and normalized to total STAT1. (C) Whole cell lysates were prepared and analyzed via western blot by using antibodies directed against phosphorylated-STAT3, total STAT3, and GAPDH. Mean phosphorylated-STAT3 levels were determined using densitometric analysis and normalized to total STAT3. The data are presented as means ± SEM of three independent experiments. Asterisks (* and **) indicate responses that are significantly different (p

Techniques Used: Derivative Assay, Western Blot

16) Product Images from "NEMO Peptide Inhibits the Growth of Pancreatic Ductal Adenocarcinoma by Blocking NF-κB Activation"

Article Title: NEMO Peptide Inhibits the Growth of Pancreatic Ductal Adenocarcinoma by Blocking NF-κB Activation

Journal: Cancer letters

doi: 10.1016/j.canlet.2017.09.018

A. Inhibition of NF-κB downstream target genes by NBDP and gemcitabine. Immunohistochemical analysis of HPNE/Kras G12V /P16sh tumors and normal pancreatic tissues stained with antibodies to Ki67, cyclin D1, phosphorylated ERK, and the active P65 subunit of NF-κB; B. C . Levels of Ki67 and cyclin D1 expression on immunohistochemical analysis were decreased in tumor tissue treated with NBDP combined with gemcitabine compared with the control, NBDP, and gemcitabine (Ki67: combination vs gemcitabine, *P=0.0345; cyclin D1: combination vs gemcitabine, *P=0.0118); D,E . Levels of phosphorylated Erk and phosphorylated P65 expression in tumor tissue treated with gemcitabine on immunohistochemical analysis were increased compared with the control but were decreased in tumor tissue treated with NBDP combined with gemcitabine compared with the control and gemcitabine alone (P-Erk: gemcitabine vs control, *P=0.0386, combination vs gemcitabine, **P=0.0172; P-P65: gemcitabine vs control, *P=0.0263, combination vs gemcitabine, **P=0.0080); F . Western blot analysis showed the expression of phosphorylated P65 and P65 in a total cell lysate of HPNE/Kras G12V /P16sh tumor tissue in each group; G . Phosphorylated P65 expression of HPNE/Kras G12V /P16sh tumor tissue treated with NBDP combined with gemcitabine was significantly down-regulated compared with that in the control and gemcitabine groups (gemcitabine vs control, *P=0.0479, combination vs gemcitabine, **P=0.0043). Ctrl: control; Gem: gemcitabine.
Figure Legend Snippet: A. Inhibition of NF-κB downstream target genes by NBDP and gemcitabine. Immunohistochemical analysis of HPNE/Kras G12V /P16sh tumors and normal pancreatic tissues stained with antibodies to Ki67, cyclin D1, phosphorylated ERK, and the active P65 subunit of NF-κB; B. C . Levels of Ki67 and cyclin D1 expression on immunohistochemical analysis were decreased in tumor tissue treated with NBDP combined with gemcitabine compared with the control, NBDP, and gemcitabine (Ki67: combination vs gemcitabine, *P=0.0345; cyclin D1: combination vs gemcitabine, *P=0.0118); D,E . Levels of phosphorylated Erk and phosphorylated P65 expression in tumor tissue treated with gemcitabine on immunohistochemical analysis were increased compared with the control but were decreased in tumor tissue treated with NBDP combined with gemcitabine compared with the control and gemcitabine alone (P-Erk: gemcitabine vs control, *P=0.0386, combination vs gemcitabine, **P=0.0172; P-P65: gemcitabine vs control, *P=0.0263, combination vs gemcitabine, **P=0.0080); F . Western blot analysis showed the expression of phosphorylated P65 and P65 in a total cell lysate of HPNE/Kras G12V /P16sh tumor tissue in each group; G . Phosphorylated P65 expression of HPNE/Kras G12V /P16sh tumor tissue treated with NBDP combined with gemcitabine was significantly down-regulated compared with that in the control and gemcitabine groups (gemcitabine vs control, *P=0.0479, combination vs gemcitabine, **P=0.0043). Ctrl: control; Gem: gemcitabine.

Techniques Used: Inhibition, Immunohistochemistry, Staining, Expressing, Western Blot

17) Product Images from "Targeting PML-RARα and Oncogenic Signaling Pathways by Chinese Herbal Mixture Tien-Hsien Liquid in Acute Promyelocytic Leukemia NB4 Cells"

Article Title: Targeting PML-RARα and Oncogenic Signaling Pathways by Chinese Herbal Mixture Tien-Hsien Liquid in Acute Promyelocytic Leukemia NB4 Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1093/ecam/nep165

THL-induced inhibition of oncogenic signaling pathways in NB4 cells. Western blot analysis for phosphorylated (p-) and total Akt (a), mTOR (b), Stat3 (c) and ERK (d) proteins of THL-treated NB4 cells. Phosphorylated Akt, mTOR, Stat3 and ERK were all significantly inhibited by treatment with THL. Total protein levels of Akt, mTOR and Stat3 were also significantly decreased by THL, while the total ERK protein was only slightly decreased by the highest dose of THL. The cells were treated with PBS or THL (0.375–3 mg/ml) for 72 h and then harvested. Cell lysates were then prepared for western blot analysis.
Figure Legend Snippet: THL-induced inhibition of oncogenic signaling pathways in NB4 cells. Western blot analysis for phosphorylated (p-) and total Akt (a), mTOR (b), Stat3 (c) and ERK (d) proteins of THL-treated NB4 cells. Phosphorylated Akt, mTOR, Stat3 and ERK were all significantly inhibited by treatment with THL. Total protein levels of Akt, mTOR and Stat3 were also significantly decreased by THL, while the total ERK protein was only slightly decreased by the highest dose of THL. The cells were treated with PBS or THL (0.375–3 mg/ml) for 72 h and then harvested. Cell lysates were then prepared for western blot analysis.

Techniques Used: Inhibition, Western Blot

18) Product Images from "Bajijiasu Abrogates Osteoclast Differentiation via the Suppression of RANKL Signaling Pathways through NF-κB and NFAT"

Article Title: Bajijiasu Abrogates Osteoclast Differentiation via the Suppression of RANKL Signaling Pathways through NF-κB and NFAT

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010203

Bajijiasu suppresses RANKL-induced NF-κB activity and proteins of IκB-α and phosphorylation of extracellular signal-regulated kinases (ERK). ( A ) Luciferase activity in RANKL stimulated NF-κB transfected RAW264.7 cells under an luciferase construct. Cells were added with Bajijiasu and then stimulated with glutathione S -transferase (GST)-rRANKL (50 ng/mL). (* p
Figure Legend Snippet: Bajijiasu suppresses RANKL-induced NF-κB activity and proteins of IκB-α and phosphorylation of extracellular signal-regulated kinases (ERK). ( A ) Luciferase activity in RANKL stimulated NF-κB transfected RAW264.7 cells under an luciferase construct. Cells were added with Bajijiasu and then stimulated with glutathione S -transferase (GST)-rRANKL (50 ng/mL). (* p

Techniques Used: Activity Assay, Luciferase, Transfection, Construct

19) Product Images from "ERK/GSK3\u00df/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition"

Article Title: ERK/GSK3\u00df/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2011.11.024

Schematic representation of the proposed mechanism for radiation-induced EMT in alveolar type II epithelial cells. Radiation-induced generation of ROS leads to activation of the MEK/ERK signaling pathway. Activated ERK1/2 causes the inactivation of GSK3ß
Figure Legend Snippet: Schematic representation of the proposed mechanism for radiation-induced EMT in alveolar type II epithelial cells. Radiation-induced generation of ROS leads to activation of the MEK/ERK signaling pathway. Activated ERK1/2 causes the inactivation of GSK3ß

Techniques Used: Activation Assay

Pre-incubating RLE-6TN cells with NAC abrogates the radiation-induced changes in p -ERK, Snail α-SMA and E-cadherin. RLE-6TN cells were preincubated with 5 mM NAC for 3 h prior to radiation with 8Gy. Intracellular ROS generation was measured using
Figure Legend Snippet: Pre-incubating RLE-6TN cells with NAC abrogates the radiation-induced changes in p -ERK, Snail α-SMA and E-cadherin. RLE-6TN cells were preincubated with 5 mM NAC for 3 h prior to radiation with 8Gy. Intracellular ROS generation was measured using

Techniques Used:

Inhibition of ERK abolishes the radiation-induced changes in α-SMA, E-cadherin, p -GSK3ß and Snail. RLE-6TN cells were incubated with/without U0126 for 2 h prior to irradiation with 8 Gy of 137 Cs γ rays. Cell lysates were collected
Figure Legend Snippet: Inhibition of ERK abolishes the radiation-induced changes in α-SMA, E-cadherin, p -GSK3ß and Snail. RLE-6TN cells were incubated with/without U0126 for 2 h prior to irradiation with 8 Gy of 137 Cs γ rays. Cell lysates were collected

Techniques Used: Inhibition, Incubation, Irradiation

20) Product Images from "Central Role of Gq in the Hypertrophic Signal Transduction of Angiotensin II in Vascular Smooth Muscle Cells"

Article Title: Central Role of Gq in the Hypertrophic Signal Transduction of Angiotensin II in Vascular Smooth Muscle Cells

Journal: Endocrinology

doi: 10.1210/en.2007-1694

G q I selectively blocks the EGFR/ERK cascade induced by AngII. A and B, VSMCs infected with adenovirus encoding G q I minigene or control vector (20 moi) were stimulated with AngII (100 n m ) for 2 min (A) or 10 min (B) and immunoblotted with antibodies against
Figure Legend Snippet: G q I selectively blocks the EGFR/ERK cascade induced by AngII. A and B, VSMCs infected with adenovirus encoding G q I minigene or control vector (20 moi) were stimulated with AngII (100 n m ) for 2 min (A) or 10 min (B) and immunoblotted with antibodies against

Techniques Used: Infection, Plasmid Preparation

The EGFR/ERK cascade induced by AngII was blocked by YM-254890, a pharmacological G q inhibitor. A, VSMCs pretreated with vehicle (dimethylsulfoxide 0.1%) or YM-254890 (1 μ m ) for 10 min were stimulated with AngII (100 n m ). Intracellular Ca 2+
Figure Legend Snippet: The EGFR/ERK cascade induced by AngII was blocked by YM-254890, a pharmacological G q inhibitor. A, VSMCs pretreated with vehicle (dimethylsulfoxide 0.1%) or YM-254890 (1 μ m ) for 10 min were stimulated with AngII (100 n m ). Intracellular Ca 2+

Techniques Used:

21) Product Images from "Chelidonic acid evokes antidepressant-like effect through the up-regulation of BDNF in forced swimming test"

Article Title: Chelidonic acid evokes antidepressant-like effect through the up-regulation of BDNF in forced swimming test

Journal: Experimental Biology and Medicine

doi: 10.1177/1535370216642044

Effect of CA on the levels of BDNF and pERK in the hippocampus. (a) The levels of BDNF were analyzed by Western blot analysis. (b) Hippocampal tissues were stained with DAB (for BDNF, arrow). (c) Phosphorylated ERK was analyzed by Western blot analysis. Protein levels were quantified by densitometry. Values are means ± SEM (A and C lower panels). * P
Figure Legend Snippet: Effect of CA on the levels of BDNF and pERK in the hippocampus. (a) The levels of BDNF were analyzed by Western blot analysis. (b) Hippocampal tissues were stained with DAB (for BDNF, arrow). (c) Phosphorylated ERK was analyzed by Western blot analysis. Protein levels were quantified by densitometry. Values are means ± SEM (A and C lower panels). * P

Techniques Used: Western Blot, Staining

22) Product Images from "Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis"

Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

Journal: Technology in Cancer Research & Treatment

doi: 10.1177/1533034617754024

Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
Figure Legend Snippet: Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

Techniques Used:

Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.
Figure Legend Snippet: Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

Techniques Used: Expressing

Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.
Figure Legend Snippet: Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

Techniques Used: Immunohistochemistry

23) Product Images from "DUSP6, a tumor suppressor, is involved in differentiation and apoptosis in esophageal squamous cell carcinoma"

Article Title: DUSP6, a tumor suppressor, is involved in differentiation and apoptosis in esophageal squamous cell carcinoma

Journal: Oncology Letters

doi: 10.3892/ol.2013.1605

DUSP6 expression in esophageal cancer. (A) Expression of DUSP6 in normal esophageal epithelia and primary ESCC tumors were examined by immunohistochemistry. Normal esophageal epithelia are shown in panel N and the primary esophageal cancers are shown in panels T1, T2, and T3 (magnification: Left, ×40; right, ×100). (B) Expression of DUSP6 in paired normal and tumor tissues from the same patients by qPCR. In total, 36.8% (7/19) of the biopsies displayed at least two-fold downregulation of DUSP6 compared with their corresponding normal counterparts. The dotted line is shown to indicate the two-fold threshold of downregulation. (C) Western blot analysis was used to determine the DUSP6 expression in EC9706, empty vector-transfected EC9706, pCMV-DUSP6 (DUSP6)-transfected EC9706, KYSE150, empty vector-transfected KYSE150 and pCMV-DUSP6-transfected KYSE150 cells. (D) Western blot analysis was utilized to examine the DUSP6 and p-ERK expression in EC9706, empty vector-transfected EC9706 and pCMV-DUSP6 (DUSP6)-transfected EC9706 cells. DUSP6, dual-specificity phosphatase 6; ESCC, esophageal squamous cell carcinoma; p-ERK, phosphorylated extracellular signal-regulated kinase.
Figure Legend Snippet: DUSP6 expression in esophageal cancer. (A) Expression of DUSP6 in normal esophageal epithelia and primary ESCC tumors were examined by immunohistochemistry. Normal esophageal epithelia are shown in panel N and the primary esophageal cancers are shown in panels T1, T2, and T3 (magnification: Left, ×40; right, ×100). (B) Expression of DUSP6 in paired normal and tumor tissues from the same patients by qPCR. In total, 36.8% (7/19) of the biopsies displayed at least two-fold downregulation of DUSP6 compared with their corresponding normal counterparts. The dotted line is shown to indicate the two-fold threshold of downregulation. (C) Western blot analysis was used to determine the DUSP6 expression in EC9706, empty vector-transfected EC9706, pCMV-DUSP6 (DUSP6)-transfected EC9706, KYSE150, empty vector-transfected KYSE150 and pCMV-DUSP6-transfected KYSE150 cells. (D) Western blot analysis was utilized to examine the DUSP6 and p-ERK expression in EC9706, empty vector-transfected EC9706 and pCMV-DUSP6 (DUSP6)-transfected EC9706 cells. DUSP6, dual-specificity phosphatase 6; ESCC, esophageal squamous cell carcinoma; p-ERK, phosphorylated extracellular signal-regulated kinase.

Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection

24) Product Images from "Prostaglandin E2 stimulates COX-2 expression via mitogen-activated protein kinase p38 but not ERK in human follicular dendritic cell-like cells"

Article Title: Prostaglandin E2 stimulates COX-2 expression via mitogen-activated protein kinase p38 but not ERK in human follicular dendritic cell-like cells

Journal: BMC Immunology

doi: 10.1186/s12865-020-00347-y

IL-1β induces phosphorylation of both ERK and p38 in FDC-like cells. The total and phosphorylated levels of ERK ( a ) and p38 ( b ) MAPKs were examined by immunoblotting before and at the indicated time points after culture of FDC-like cells in the presence of IL-1β (10 pg/ml). Representative immunoblots and statistical analysis data (mean ± SEM) of three ( a ) or two ( b ) independent experiments are depicted. An asterisk(s) indicates a significant difference (*, p
Figure Legend Snippet: IL-1β induces phosphorylation of both ERK and p38 in FDC-like cells. The total and phosphorylated levels of ERK ( a ) and p38 ( b ) MAPKs were examined by immunoblotting before and at the indicated time points after culture of FDC-like cells in the presence of IL-1β (10 pg/ml). Representative immunoblots and statistical analysis data (mean ± SEM) of three ( a ) or two ( b ) independent experiments are depicted. An asterisk(s) indicates a significant difference (*, p

Techniques Used: Western Blot

IL-1β-stimulated COX-2 expression in FDC-like cells depends on p38 MAPK. a The effects of ERK and p38 knockdown on COX-2 induction by IL-1β were examined with FDC-like cells that had been transfected with control or siRNA against ERK or p38 before the addition of IL-1β (10 pg/ml). The expression levels of COX-1, COX-2, and β-actin were measured by immunoblotting. b The amounts of 6-keto-PGF 1α in culture supernatants of (A) experiments were measured by EIA as described in Methods . c The effects of PD098059 (PD, 50 μM) and SB203580 (SB, 10 μM) on COX-2 induction by IL-1β were examined. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (*, p
Figure Legend Snippet: IL-1β-stimulated COX-2 expression in FDC-like cells depends on p38 MAPK. a The effects of ERK and p38 knockdown on COX-2 induction by IL-1β were examined with FDC-like cells that had been transfected with control or siRNA against ERK or p38 before the addition of IL-1β (10 pg/ml). The expression levels of COX-1, COX-2, and β-actin were measured by immunoblotting. b The amounts of 6-keto-PGF 1α in culture supernatants of (A) experiments were measured by EIA as described in Methods . c The effects of PD098059 (PD, 50 μM) and SB203580 (SB, 10 μM) on COX-2 induction by IL-1β were examined. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (*, p

Techniques Used: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

p38 MAPK is necessary for the PGE 2 -induced COX-2 expression in FDC-like cells. a The effects of ERK and p38 knockdown on COX-2 induction by PGE 2 were examined with FDC-like cells that had been transfected with control or siRNA against ERK or p38 before the addition of PGE 2 (1 μM). b The effects of PD098059 (PD, 50 μM) and SB203580 (SB, 10 μM) on COX-2 induction by PGE 2 were examined. FDC-like cells (1 × 10 5 cells) were cultured in the presence of PD098059 or SB203580 for 30 min and then added with PGE 2 (1 μM) for 8 h. The expression levels of COX-1, COX-2, and β-actin were measured by immunoblotting. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (*, p
Figure Legend Snippet: p38 MAPK is necessary for the PGE 2 -induced COX-2 expression in FDC-like cells. a The effects of ERK and p38 knockdown on COX-2 induction by PGE 2 were examined with FDC-like cells that had been transfected with control or siRNA against ERK or p38 before the addition of PGE 2 (1 μM). b The effects of PD098059 (PD, 50 μM) and SB203580 (SB, 10 μM) on COX-2 induction by PGE 2 were examined. FDC-like cells (1 × 10 5 cells) were cultured in the presence of PD098059 or SB203580 for 30 min and then added with PGE 2 (1 μM) for 8 h. The expression levels of COX-1, COX-2, and β-actin were measured by immunoblotting. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (*, p

Techniques Used: Expressing, Transfection, Cell Culture, Western Blot

PGE 2 selectively increases phosphorylation of p38 in FDC-like cells. The total and phosphorylated levels of ERK ( a ) and p38 ( b ) MAPKs were examined by immunoblotting before and at the indicated time points after culture of FDC-like cells in the presence or absence of PGE 2 (1 μM). β-Actin was used to show equal loading of cell lysates. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (**, p
Figure Legend Snippet: PGE 2 selectively increases phosphorylation of p38 in FDC-like cells. The total and phosphorylated levels of ERK ( a ) and p38 ( b ) MAPKs were examined by immunoblotting before and at the indicated time points after culture of FDC-like cells in the presence or absence of PGE 2 (1 μM). β-Actin was used to show equal loading of cell lysates. Representative immunoblots and statistical analysis data (mean ± SEM) of three independent experiments are shown. An asterisk(s) indicates a significant difference (**, p

Techniques Used: Western Blot

25) Product Images from "The Critical Role of Membrane Cholesterol in Salmonella-Induced Autophagy in Intestinal Epithelial Cells"

Article Title: The Critical Role of Membrane Cholesterol in Salmonella-Induced Autophagy in Intestinal Epithelial Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms150712558

The role of ERK or Akt on the Beclin 1 and LC3II proteins expression in Salmonella -infected Caco-2 cells. Caco-2 cells were uninfected (Con) or infected by wild-type S . typhimurium strain SL1344 for indicated times, in the presence or absence of PD98059 (PD) or LY294002 (LY). Immunoblots were performed on whole cell lysates with antibody to detect Beclin 1 and LC3II expression, or GAPDH for normalization of proteins. Representative immunoblots of Beclin 1 and LC3II proteins expression in Salmonella -infected Caco-2 cells in the presence of PD ( A ) or LY ( B ) are shown.
Figure Legend Snippet: The role of ERK or Akt on the Beclin 1 and LC3II proteins expression in Salmonella -infected Caco-2 cells. Caco-2 cells were uninfected (Con) or infected by wild-type S . typhimurium strain SL1344 for indicated times, in the presence or absence of PD98059 (PD) or LY294002 (LY). Immunoblots were performed on whole cell lysates with antibody to detect Beclin 1 and LC3II expression, or GAPDH for normalization of proteins. Representative immunoblots of Beclin 1 and LC3II proteins expression in Salmonella -infected Caco-2 cells in the presence of PD ( A ) or LY ( B ) are shown.

Techniques Used: Expressing, Infection, Western Blot

26) Product Images from "Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells"

Article Title: Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-28-28

Effects of PIA treatment on Akt and Akt-related signaling molecules . (A) P-Akt level in KB and KOSCC-25B cells was significantly lower after 5 μM PIA treatment for 24 hours. However, Akt1/2 and ILK (upstream molecules of Akt) did not show any change after PIA treatment. (B) Inhibition of Akt activity by PIA induced downregulation of p50 and p-p65 in KB and KOSCC-25B cells, but it did not affect phosphorylation of JNK, p38, and ERK.
Figure Legend Snippet: Effects of PIA treatment on Akt and Akt-related signaling molecules . (A) P-Akt level in KB and KOSCC-25B cells was significantly lower after 5 μM PIA treatment for 24 hours. However, Akt1/2 and ILK (upstream molecules of Akt) did not show any change after PIA treatment. (B) Inhibition of Akt activity by PIA induced downregulation of p50 and p-p65 in KB and KOSCC-25B cells, but it did not affect phosphorylation of JNK, p38, and ERK.

Techniques Used: Inhibition, Activity Assay

27) Product Images from "Biflorin Ameliorates Memory Impairments Induced by Cholinergic Blockade in Mice"

Article Title: Biflorin Ameliorates Memory Impairments Induced by Cholinergic Blockade in Mice

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2016.058

Effects of biflorin on memory-related proteins in the hippocampus. The mice were administered biflorin (0.3 or 1 mg/kg, p.o.) or the same volume of vehicle (10% Tween 80 solution) and sacrificed 1 h after drug administration. The immunoreactivity and quantitative analysis of PKC-ζ, phosphorylated PKC (pPKC-ζ) (A), CaMKII, phosphorylated CaMKII (pCaMKII) (B), ERK, phosphorylated ERK (pERK) (C), CREB, and phosphorylated CREB (pCREB) (D) were measured in the hippocampal tissue. Data represent the means ± SEM (n=3-4/group) ( * p
Figure Legend Snippet: Effects of biflorin on memory-related proteins in the hippocampus. The mice were administered biflorin (0.3 or 1 mg/kg, p.o.) or the same volume of vehicle (10% Tween 80 solution) and sacrificed 1 h after drug administration. The immunoreactivity and quantitative analysis of PKC-ζ, phosphorylated PKC (pPKC-ζ) (A), CaMKII, phosphorylated CaMKII (pCaMKII) (B), ERK, phosphorylated ERK (pERK) (C), CREB, and phosphorylated CREB (pCREB) (D) were measured in the hippocampal tissue. Data represent the means ± SEM (n=3-4/group) ( * p

Techniques Used: Mouse Assay

28) Product Images from "Functional characterization of chimpanzee cytomegalovirus chemokine, vCXCL-1CCMV"

Article Title: Functional characterization of chimpanzee cytomegalovirus chemokine, vCXCL-1CCMV

Journal:

doi: 10.1016/j.virol.2007.03.002

Both viral and host chemokines activate the ERK and Akt pathways. Differentiated HL-60 cells stably expressing CXCR2 were stimulated with 100 nM chemokine for 1 min at room temperature and lysed instantly. Lysates were immunoblotted using antibodies against
Figure Legend Snippet: Both viral and host chemokines activate the ERK and Akt pathways. Differentiated HL-60 cells stably expressing CXCR2 were stimulated with 100 nM chemokine for 1 min at room temperature and lysed instantly. Lysates were immunoblotted using antibodies against

Techniques Used: Stable Transfection, Expressing

29) Product Images from "4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression"

Article Title: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2172-2

Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis
Figure Legend Snippet: Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

Techniques Used: Activation Assay, Concentration Assay, Western Blot

30) Product Images from "Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells"

Article Title: Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells

Journal: Food and Chemical Toxicology

doi: 10.1016/j.fct.2011.10.031

Effect of CdCl 2 treatment on pERK activation in Saos-2 cells. (A) Representative western blot of pERK and ERK at 3, 4, or 18hr in (C) control or cells treated with (T) 10 μM CdCl 2 .Controls received culture medium only. (B) Relative density of pERK/ERK measured by densitometry. Each bar represents the mean ±SEM of three independent experiments.* denotes significant from control p
Figure Legend Snippet: Effect of CdCl 2 treatment on pERK activation in Saos-2 cells. (A) Representative western blot of pERK and ERK at 3, 4, or 18hr in (C) control or cells treated with (T) 10 μM CdCl 2 .Controls received culture medium only. (B) Relative density of pERK/ERK measured by densitometry. Each bar represents the mean ±SEM of three independent experiments.* denotes significant from control p

Techniques Used: Activation Assay, Western Blot

31) Product Images from "Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw"

Article Title: Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw

Journal:

doi: 10.1038/sj.bjp.0707270

Effect of α -humulene or trans -caryophyllene on LPS-induced MAPK activation. Time-dependent activation of ( a ) ERK, ( b ) p38 MAPK and ( c ) JNK. Rats were treated with saline or LPS (1 μ g per paw) and then the paws were isolated at
Figure Legend Snippet: Effect of α -humulene or trans -caryophyllene on LPS-induced MAPK activation. Time-dependent activation of ( a ) ERK, ( b ) p38 MAPK and ( c ) JNK. Rats were treated with saline or LPS (1 μ g per paw) and then the paws were isolated at

Techniques Used: Activation Assay, Isolation

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Western Blot:

Article Title: Foxo3a-mediated overexpression of microRNA-622 suppresses tumor metastasis by repressing hypoxia-inducible factor-1α in erk-responsive lung cancer
Article Snippet: .. Western blotting used primary antibodies (diluted 1:1000) against the following proteins: AKT (sc-8312), phosphorylated AKT (sc-16646-R), ERK (sc-94), phosphorylated ERK (sc-7383), and vimentin (sc-6260); each antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). .. Antibodies against HIF-1α (Cell Signaling) and E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ) were also used.

Incubation:

Article Title: Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines
Article Snippet: .. Membranes were blocked in TTBS containing 5% nonfat dry milk for 1 h then incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, CA, USA) for phosphorylated ERK (pERK) or total ERK followed by 2 h incubation at room temperature with HRP antibody (Santa Cruz Biotechnology, CA, USA). .. Immunoreactive proteins were detected by exposing the membranes to Immun-Star HRP chemiluminescent (Bio-Rad, CA, USA), visualized using Quantity One 1-D Analysis software, and quantified using Image J software.

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    Santa Cruz Biotechnology anti phospho erk
    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.
    Anti Phospho Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho erk/product/Santa Cruz Biotechnology
    Average 93 stars, based on 50 article reviews
    Price from $9.99 to $1999.99
    anti phospho erk - by Bioz Stars, 2020-12
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    89
    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
    https://www.bioz.com/result/phospho extracellular signal regulated kinase/product/Santa Cruz Biotechnology
    Average 89 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    phospho extracellular signal regulated kinase - by Bioz Stars, 2020-12
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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 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

    Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.

    Journal: The EMBO Journal

    Article Title: MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts

    doi: 10.1093/emboj/cdg322

    Figure Lengend Snippet: Fig. 7. Fibroblast migration and calpain activity are dependent on MEK, but not JNK activity. ( A ) Wild-type fibroblasts were loaded into Transwell migration chambers (10 5 cells/well) and allowed to migrate for 5 h, using 5% serum as a chemotactic agent. Calpain inhibitor PD150606 (50 µM), MEK inhibitor UO126 (10 µM) or matrix metalloproteinase inhibitor GM6001 (2 µM) were added to both the upper and lower chambers of the designated wells. ( B ) Wild-type fibroblasts were pre-incubated for 1 h with JNK inhibitor SP600125 (10 µM) or MEK inhibitor UO126 (10 µM), and calpain activity was assessed by SLLVY-AMC cleavage, and compared with that of non-treated cells. The results of both (A) and (B) are the mean ± SEM of at least three independent experiments. ( C ) Wild-type fibroblasts were pre-incubated with 10 µM SP600125 or 10 µM UO126 for 1 h and then analyzed for migration using the in vitro wound healing assay in the continuous presence of inhibitor. ( D ) Serum-starved wild-type, MEKK1–/– or MEKK1 add-back fibroblasts were treated with EGF or FGF-2 for 10 min and then lysed. ERK1/2 activation was then assessed by phospho-ERK immunoblotting. The membrane was then stripped and the total ERK2 level determined by ERK2 immunoblotting. The data are representative of at least three independent experiments. NS, no stimulus.

    Article Snippet: Anti-phospho ERK, ERK2 and FAK (C-terminal) antibodies used for immunoblotting were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Migration, Activity Assay, Incubation, In Vitro, Wound Healing Assay, Activation 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