Structured Review

Cell Signaling Technology Inc anti erk
MKP-1 inhibited APP processing through inactivation of the <t>ERK/MAPK</t> signaling pathway. a – e Immunoblot of the expression of MKP-1 b , P-ERK c , <t>P-JNK</t> d and P-P38 e in lysates of N2A APP cells after overexpression of MKP-1 by LV MKP-1 or knockdown of MKP-1 by LV shMKP-1 . * p
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1) Product Images from "MKP-1 reduces Aβ generation and alleviates cognitive impairments in Alzheimer’s disease models"

Article Title: MKP-1 reduces Aβ generation and alleviates cognitive impairments in Alzheimer’s disease models

Journal: Signal Transduction and Targeted Therapy

doi: 10.1038/s41392-019-0091-4

MKP-1 inhibited APP processing through inactivation of the ERK/MAPK signaling pathway. a – e Immunoblot of the expression of MKP-1 b , P-ERK c , P-JNK d and P-P38 e in lysates of N2A APP cells after overexpression of MKP-1 by LV MKP-1 or knockdown of MKP-1 by LV shMKP-1 . * p
Figure Legend Snippet: MKP-1 inhibited APP processing through inactivation of the ERK/MAPK signaling pathway. a – e Immunoblot of the expression of MKP-1 b , P-ERK c , P-JNK d and P-P38 e in lysates of N2A APP cells after overexpression of MKP-1 by LV MKP-1 or knockdown of MKP-1 by LV shMKP-1 . * p

Techniques Used: Expressing, Over Expression

2) Product Images from "Inhibition of protein kinase II (CK2) prevents induced signal transducer and activator of transcription (STAT) 1/3 and constitutive STAT3 activation"

Article Title: Inhibition of protein kinase II (CK2) prevents induced signal transducer and activator of transcription (STAT) 1/3 and constitutive STAT3 activation

Journal: Oncotarget

doi:

Inhibition of CK2 blocks ERK and Akt signaling (A) HepG2 cells were stimulated with 10 ng/ml Hyper-IL-6, and cells were harvested after 0, 15, 30 and 60min. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation and expression level of Akt were assessed by Western blotting, and β-Actin served as internal loading control. (B, C) Ba/F3-gp130 cells were stimulated with 10 ng/ml Hyper-IL-6 or 10 ng/ml IL-27. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation of Akt was assessed by Western blotting. (D) The experiment was performed as described under panel C, but Ba/F3-gp130-LIFR cells were stimulated with 10 ng/ml CT-1. (E-G) The experiments were performed as described under panel B-D. Phosphorylation and expression level of ERK were assessed by Western blotting, and β-actin served as internal loading control. (H) HepG2 cells were stimulated with 10 ng/ml Hyper-IL-6, and cells were harvested after 0, 15, 30 and 60 min. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation and expression levels of ERK were assessed by Western blotting, and β-actin served as internal loading control. One representative Western blot from at least three independent experiments is shown.
Figure Legend Snippet: Inhibition of CK2 blocks ERK and Akt signaling (A) HepG2 cells were stimulated with 10 ng/ml Hyper-IL-6, and cells were harvested after 0, 15, 30 and 60min. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation and expression level of Akt were assessed by Western blotting, and β-Actin served as internal loading control. (B, C) Ba/F3-gp130 cells were stimulated with 10 ng/ml Hyper-IL-6 or 10 ng/ml IL-27. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation of Akt was assessed by Western blotting. (D) The experiment was performed as described under panel C, but Ba/F3-gp130-LIFR cells were stimulated with 10 ng/ml CT-1. (E-G) The experiments were performed as described under panel B-D. Phosphorylation and expression level of ERK were assessed by Western blotting, and β-actin served as internal loading control. (H) HepG2 cells were stimulated with 10 ng/ml Hyper-IL-6, and cells were harvested after 0, 15, 30 and 60 min. Cells were either pre-treated with 100 μM TBB for 90 min or with DMSO as control. Phosphorylation and expression levels of ERK were assessed by Western blotting, and β-actin served as internal loading control. One representative Western blot from at least three independent experiments is shown.

Techniques Used: Inhibition, Expressing, Western Blot

CK2 is the central lynchpin of the signaling pathways investigated in this study The schematic overview shows that IL-6 family cytokines activate the three major signaling pathways Jak/STAT, Ras/Raf/MEK/ERK, and Akt/PI3K. All of them need CK2 activity for the initiation of downstream signaling. The gp130ΔYY-mutant, which is constitutively active and signals from the cell membrane and from intracellular compartments, solely activates Jak/STAT signaling, and this can be efficiently blocked through CK2 blockade. A constitutively active STAT3 mutant (STAT3Y640F), which is constitutively phosphorylated by Jak1 and Src kinase, also needs CK2 activity
Figure Legend Snippet: CK2 is the central lynchpin of the signaling pathways investigated in this study The schematic overview shows that IL-6 family cytokines activate the three major signaling pathways Jak/STAT, Ras/Raf/MEK/ERK, and Akt/PI3K. All of them need CK2 activity for the initiation of downstream signaling. The gp130ΔYY-mutant, which is constitutively active and signals from the cell membrane and from intracellular compartments, solely activates Jak/STAT signaling, and this can be efficiently blocked through CK2 blockade. A constitutively active STAT3 mutant (STAT3Y640F), which is constitutively phosphorylated by Jak1 and Src kinase, also needs CK2 activity

Techniques Used: Activity Assay, Mutagenesis

3) Product Images from "Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2"

Article Title: Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2011.01532.x

(A) Plumbagin inhibited VEGF-induced MEK, ERK and JNK phosphorylation using Western blotting analysis; 40 ng protein of the whole cell lysate was loaded and run on 10–12% SDS-PAGE. Phosphorylated MEK, phosphorylated ERK and phosphorylated JNK were detected by specific antibodies. The grey analysis was shown in digital format located between the Western blot bands of total and activated forms of proteins. (B) Proposed model for how plumbagin represses tumour angiogenesis through Ras signalling pathways in endothelial cells. Based on the inhibition of VEGFR2 activation, plumbagin may inhibit endothelial cell migration by affecting Ras downstream cascade of the Ras–Rac–cofilin pathway. In addition, plumbagin may suppress endothelial cell growth by acting on the Ras-mediated cascade of the Ras/MEK/ERK and Ras/MEK /JNK pathways.
Figure Legend Snippet: (A) Plumbagin inhibited VEGF-induced MEK, ERK and JNK phosphorylation using Western blotting analysis; 40 ng protein of the whole cell lysate was loaded and run on 10–12% SDS-PAGE. Phosphorylated MEK, phosphorylated ERK and phosphorylated JNK were detected by specific antibodies. The grey analysis was shown in digital format located between the Western blot bands of total and activated forms of proteins. (B) Proposed model for how plumbagin represses tumour angiogenesis through Ras signalling pathways in endothelial cells. Based on the inhibition of VEGFR2 activation, plumbagin may inhibit endothelial cell migration by affecting Ras downstream cascade of the Ras–Rac–cofilin pathway. In addition, plumbagin may suppress endothelial cell growth by acting on the Ras-mediated cascade of the Ras/MEK/ERK and Ras/MEK /JNK pathways.

Techniques Used: Western Blot, SDS Page, Inhibition, Activation Assay, Migration

4) Product Images from "CpG-ODN, the TLR9 agonist, attenuates myocardial ischemia/reperfusion injury: involving activation of PI3K/Akt signaling"

Article Title: CpG-ODN, the TLR9 agonist, attenuates myocardial ischemia/reperfusion injury: involving activation of PI3K/Akt signaling

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbadis.2012.08.008

CpG-ODN treatment increased the levels of phosphorylated Akt, GSK-3β and ERK in the myocardium. Mice were treated with or without CpG-ODN one hr prior to myocardial I/R (n=6/group). Sham operated mice served as sham control (n=4). A PI3K inhibitor,
Figure Legend Snippet: CpG-ODN treatment increased the levels of phosphorylated Akt, GSK-3β and ERK in the myocardium. Mice were treated with or without CpG-ODN one hr prior to myocardial I/R (n=6/group). Sham operated mice served as sham control (n=4). A PI3K inhibitor,

Techniques Used: Mouse Assay

5) Product Images from "Pituitary Adenylate Cyclase-Activating Polypeptide Causes Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor in Lung Cancer Cells"

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Causes Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.111.190033

PACAP-27 increases EGFR and ERK tyrosine phosphorylation in a time- and dose-dependent manner. A, the ability of 100 nM PACAP-27 to increase EGFR tyrosine phosphorylation was investigated as a function of time by using NCI-H838 cells and antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for three determinations. C, the ability of 100 nM PACAP-27 to increase ERK tyrosine phosphorylation is indicated by using NCI-H838 cells. D, the mean percentage of ERK tyrosine phosphorylation ± S.D is indicated for four determinations. E, the ability of PACAP-27 to increase EGFR tyrosine phosphorylation is indicated as a function of dose by using NCI-H838 cells. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p
Figure Legend Snippet: PACAP-27 increases EGFR and ERK tyrosine phosphorylation in a time- and dose-dependent manner. A, the ability of 100 nM PACAP-27 to increase EGFR tyrosine phosphorylation was investigated as a function of time by using NCI-H838 cells and antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for three determinations. C, the ability of 100 nM PACAP-27 to increase ERK tyrosine phosphorylation is indicated by using NCI-H838 cells. D, the mean percentage of ERK tyrosine phosphorylation ± S.D is indicated for four determinations. E, the ability of PACAP-27 to increase EGFR tyrosine phosphorylation is indicated as a function of dose by using NCI-H838 cells. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p

Techniques Used:

6) Product Images from "Magnesium isoglycyrrhizinate protects hepatic L02 cells from ischemia/reperfusion induced injury"

Article Title: Magnesium isoglycyrrhizinate protects hepatic L02 cells from ischemia/reperfusion induced injury

Journal: International Journal of Clinical and Experimental Pathology

doi:

The expression of p-Akt, Akt, p-ERK, ERK was analyzed by Western Blot. L02 cells were incubated with 4 h of hypoxia followed by 24 h reoxygenation in the absence (-) or presence (+) of MgIG and mitoKATP channel modulator (5-HD, DE) as described in Materials
Figure Legend Snippet: The expression of p-Akt, Akt, p-ERK, ERK was analyzed by Western Blot. L02 cells were incubated with 4 h of hypoxia followed by 24 h reoxygenation in the absence (-) or presence (+) of MgIG and mitoKATP channel modulator (5-HD, DE) as described in Materials

Techniques Used: Expressing, Western Blot, Incubation

7) Product Images from "TIPE2 deficiency accelerates neointima formation by downregulating smooth muscle cell differentiation"

Article Title: TIPE2 deficiency accelerates neointima formation by downregulating smooth muscle cell differentiation

Journal: Cell Cycle

doi: 10.4161/cc.23325

Figure 5. The effects of TIPE2 on phenotypic switching, migration and proliferation of cultured VSMCs are dependent on ERK and P38 signaling pathways. ( A ) TIPE2 can inhibit the activation of ERK1/2 and P38 pathways. ( B–D ) The effects of TIPE2 on phenotypic switching, cell migration and proliferation of VSMCs are ERK1/2 and P38 dependent. Data are means ± SEM of one representative result obtained from three independent experiments. ***p
Figure Legend Snippet: Figure 5. The effects of TIPE2 on phenotypic switching, migration and proliferation of cultured VSMCs are dependent on ERK and P38 signaling pathways. ( A ) TIPE2 can inhibit the activation of ERK1/2 and P38 pathways. ( B–D ) The effects of TIPE2 on phenotypic switching, cell migration and proliferation of VSMCs are ERK1/2 and P38 dependent. Data are means ± SEM of one representative result obtained from three independent experiments. ***p

Techniques Used: Migration, Cell Culture, Activation Assay

8) Product Images from "The Ras GTPase-activating-like Protein IQGAP1 Mediates Nrf2 Protein Activation via the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (ERK) Kinase (MEK)-ERK Pathway"

Article Title: The Ras GTPase-activating-like Protein IQGAP1 Mediates Nrf2 Protein Activation via the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (ERK) Kinase (MEK)-ERK Pathway

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.444182

IQGAP1 mediates PEITC-induced MEK-ERK activation and MEK-ERK-induced ARE expression. A , HEK-293 cells transfected with pcDNA3.1 or IQGAP1 shRNA ( shIQ ) were treated with PEITC for 10 min. MEK and ERK phosphorylation was probed using anti-phospho-MEK (
Figure Legend Snippet: IQGAP1 mediates PEITC-induced MEK-ERK activation and MEK-ERK-induced ARE expression. A , HEK-293 cells transfected with pcDNA3.1 or IQGAP1 shRNA ( shIQ ) were treated with PEITC for 10 min. MEK and ERK phosphorylation was probed using anti-phospho-MEK (

Techniques Used: Activation Assay, Expressing, Transfection, shRNA

IQGAP1 mediates MEK-ERK-induced stabilization of Nrf2 by interfering with ubiquitination of Nrf2. A , HEK-293 cells were transfected as indicated, and the GFP-Nrf2 proteins were immunoprecipitated ( IP ) with anti-GFP antibody. Ubiquitination of Nrf2 was
Figure Legend Snippet: IQGAP1 mediates MEK-ERK-induced stabilization of Nrf2 by interfering with ubiquitination of Nrf2. A , HEK-293 cells were transfected as indicated, and the GFP-Nrf2 proteins were immunoprecipitated ( IP ) with anti-GFP antibody. Ubiquitination of Nrf2 was

Techniques Used: Transfection, Immunoprecipitation

IQGAP1 mediates MEK-ERK activation and ERK-Nrf2 interaction. A , HEK-293 cells and IQGAP1-knockdown HEK-293 cells were transfected with pcDNA3.1, GFP-Nrf2, HA-ERK, and DNEE-MEK plasmids. Cell lysates were immunoprecipitated ( IP ) with anti-HA antibody,
Figure Legend Snippet: IQGAP1 mediates MEK-ERK activation and ERK-Nrf2 interaction. A , HEK-293 cells and IQGAP1-knockdown HEK-293 cells were transfected with pcDNA3.1, GFP-Nrf2, HA-ERK, and DNEE-MEK plasmids. Cell lysates were immunoprecipitated ( IP ) with anti-HA antibody,

Techniques Used: Activation Assay, Transfection, Immunoprecipitation

9) Product Images from "Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma"

Article Title: Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

Journal: World Journal of Gastroenterology

doi: 10.3748/wjg.v24.i10.1120

Proteinase-activated receptor 2 mediates the activation of ERK and further induces epithelial-mesenchymal transition. A: The protein levels of ERK and p-ERK in HepG2 and SMMC-7721 cells; B: The morphological changes in HepG2 and SMMC-7721 cells after proteinase-activated receptor 2 knockdown; C and D: The protein levels of EGR-1, Snail, E-cadherin, and N-cadherin in HepG2 and SMMC-7721 cells. GAPDH was selected as an endogenous control.
Figure Legend Snippet: Proteinase-activated receptor 2 mediates the activation of ERK and further induces epithelial-mesenchymal transition. A: The protein levels of ERK and p-ERK in HepG2 and SMMC-7721 cells; B: The morphological changes in HepG2 and SMMC-7721 cells after proteinase-activated receptor 2 knockdown; C and D: The protein levels of EGR-1, Snail, E-cadherin, and N-cadherin in HepG2 and SMMC-7721 cells. GAPDH was selected as an endogenous control.

Techniques Used: Activation Assay

10) Product Images from "GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer"

Article Title: GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.24796

( A ) Cytoscape interaction network representing GSTM3–prey interactions. GSTM3–prey interactions were visualized by network edges. This analysis was performed to obtain the protein-protein interactions reported by the SysBiomics databases, in which we observed that TRAF6 interact with GSTM3. ( B ) Co-immunoprecipitation of GSTM3 and TRAF6. ( C ) Western blotting was performed for TRAF6, ERK, p-ERK, pERK, NF-κB, pNF-κB, IKBα, pIKBα, p38, pp38, JNK, pJNK, and TLR4 in the protein extracts from tumors xenotransplants in female mice. ( D ) Proportional Venn diagram of secreted proteins of CC cell lines with 264 common proteins. ( E ) Two proteins were identified in secreted proteins that can activate the TLR4 signal-pathway expressed in vitro in HSP60 and HPS70 ( F ) Western blot of TRL4 activators HSP70 and HSP60 in the proteins secreted by CC tumors, HSP60 was expressed in SiHa and HeLa tumors at day 50, and HSP70 protein expressed in SiHa tumors at 43 days and Hela tumors at 30 and 50 days.
Figure Legend Snippet: ( A ) Cytoscape interaction network representing GSTM3–prey interactions. GSTM3–prey interactions were visualized by network edges. This analysis was performed to obtain the protein-protein interactions reported by the SysBiomics databases, in which we observed that TRAF6 interact with GSTM3. ( B ) Co-immunoprecipitation of GSTM3 and TRAF6. ( C ) Western blotting was performed for TRAF6, ERK, p-ERK, pERK, NF-κB, pNF-κB, IKBα, pIKBα, p38, pp38, JNK, pJNK, and TLR4 in the protein extracts from tumors xenotransplants in female mice. ( D ) Proportional Venn diagram of secreted proteins of CC cell lines with 264 common proteins. ( E ) Two proteins were identified in secreted proteins that can activate the TLR4 signal-pathway expressed in vitro in HSP60 and HPS70 ( F ) Western blot of TRL4 activators HSP70 and HSP60 in the proteins secreted by CC tumors, HSP60 was expressed in SiHa and HeLa tumors at day 50, and HSP70 protein expressed in SiHa tumors at 43 days and Hela tumors at 30 and 50 days.

Techniques Used: Immunoprecipitation, Western Blot, Mouse Assay, In Vitro

11) Product Images from "Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation"

Article Title: Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation

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

doi: 10.1073/pnas.1721147115

Mechanistic explanation of the feedback loop between BRAF and MET. Biochemical analysis of phosphoproteins from L1.13 cell lysates treated with different inhibitors and analyzed by immunoblotting. ( A ) Treatment with increasing concentrations of TAK-632. The BRAF specific inhibitor induces dose–response MET phosphorylation, whereas P-ERK coherently decreases. ( B ) Time-course treatment of L1.13 cells with AZD-6244 (500 nM). MEK inhibition recapitulates BRAF inhibition and is followed by MET phosphorylation. ( C ) Treatment of L1.13 cells for 30 min with the Ser/Thr phosphatases inhibitor sodium fluoride, NaF (25 mM), alone or in combination with AZD-6244 (500 nM). NaF abrogates AZD-6244-induced MET phosphorylation, indicating the involvement of a Ser/Thr phosphatase in the phenomenon. ( D and E ) Treatment of L1.13 with AZD-6244 500 nM for 30 min is followed by concomitant activating dephosphorylation of PP2A phosphatase, MET Ser985 dephosphorylation (immunoprecipitates in E ). ( F and G ) Silencing of PP2A Ser/Thr phosphatase by two siRNAs (#58 and #59) targeting different sites. Scrambled siRNA was used as control (CTRL). Cells were treated with AZD-6244 (500 nM) for 30 min, and lysates were analyzed as earlier. DMSO was used as control vehicle. PP2A silencing prevents AZD-6244-induced MET Tyr1234-1235 phosphorylation ( F ) and abrogates dephosphorylation of MET Ser985 (immunoprecipitates in G ). Antibodies used for Western blots analysis: P-Y/S-MET, MET antibody recognizing Tyr1234-1235 and Ser985 in the phosphorylated state, respectively. MET, antibody against the MET β chain; P-ERK, antibody against phosphorylated ERK; ERK, antibody against the ERK protein. Vinculin or actin antibodies have been used to normalize the amount of loaded proteins.
Figure Legend Snippet: Mechanistic explanation of the feedback loop between BRAF and MET. Biochemical analysis of phosphoproteins from L1.13 cell lysates treated with different inhibitors and analyzed by immunoblotting. ( A ) Treatment with increasing concentrations of TAK-632. The BRAF specific inhibitor induces dose–response MET phosphorylation, whereas P-ERK coherently decreases. ( B ) Time-course treatment of L1.13 cells with AZD-6244 (500 nM). MEK inhibition recapitulates BRAF inhibition and is followed by MET phosphorylation. ( C ) Treatment of L1.13 cells for 30 min with the Ser/Thr phosphatases inhibitor sodium fluoride, NaF (25 mM), alone or in combination with AZD-6244 (500 nM). NaF abrogates AZD-6244-induced MET phosphorylation, indicating the involvement of a Ser/Thr phosphatase in the phenomenon. ( D and E ) Treatment of L1.13 with AZD-6244 500 nM for 30 min is followed by concomitant activating dephosphorylation of PP2A phosphatase, MET Ser985 dephosphorylation (immunoprecipitates in E ). ( F and G ) Silencing of PP2A Ser/Thr phosphatase by two siRNAs (#58 and #59) targeting different sites. Scrambled siRNA was used as control (CTRL). Cells were treated with AZD-6244 (500 nM) for 30 min, and lysates were analyzed as earlier. DMSO was used as control vehicle. PP2A silencing prevents AZD-6244-induced MET Tyr1234-1235 phosphorylation ( F ) and abrogates dephosphorylation of MET Ser985 (immunoprecipitates in G ). Antibodies used for Western blots analysis: P-Y/S-MET, MET antibody recognizing Tyr1234-1235 and Ser985 in the phosphorylated state, respectively. MET, antibody against the MET β chain; P-ERK, antibody against phosphorylated ERK; ERK, antibody against the ERK protein. Vinculin or actin antibodies have been used to normalize the amount of loaded proteins.

Techniques Used: Inhibition, De-Phosphorylation Assay, Western Blot

MET and BRAF inhibition. Immunoblotting analysis of phosphoproteins from L1.13 cells treated with MET and/or MEK inhibitors and/or PI3K-mTOR inhibitor. ( A ) Treatment with AZD-6244 (500 nM) and JNJ-33877605 (250 nM) alone or in combination for 30 min. AZD-6244 treatment is followed by MET phosphorylation. ERK is fully dephosphorylated on concomitant treatment with MET and MEK inhibitors. The increase of ERK phosphorylation after treatment with JNJ-38877605 is unexpected and possibly related to the adverse effect of the drug. ( B ) Treatment with AZD-6244 (500 nM), JNJ-33877605 (250 nM) and BEZ-235 (250 nM) alone or in combination for 30 min. AZD-6244 treatment induces MET phosphorylation. AKT activity increases when MEK is inhibited. ERK is fully dephosphorylated when the cells are treated with MEK inhibitor in combination with PI3K-mTOR inhibitor or MET inhibitor.
Figure Legend Snippet: MET and BRAF inhibition. Immunoblotting analysis of phosphoproteins from L1.13 cells treated with MET and/or MEK inhibitors and/or PI3K-mTOR inhibitor. ( A ) Treatment with AZD-6244 (500 nM) and JNJ-33877605 (250 nM) alone or in combination for 30 min. AZD-6244 treatment is followed by MET phosphorylation. ERK is fully dephosphorylated on concomitant treatment with MET and MEK inhibitors. The increase of ERK phosphorylation after treatment with JNJ-38877605 is unexpected and possibly related to the adverse effect of the drug. ( B ) Treatment with AZD-6244 (500 nM), JNJ-33877605 (250 nM) and BEZ-235 (250 nM) alone or in combination for 30 min. AZD-6244 treatment induces MET phosphorylation. AKT activity increases when MEK is inhibited. ERK is fully dephosphorylated when the cells are treated with MEK inhibitor in combination with PI3K-mTOR inhibitor or MET inhibitor.

Techniques Used: Inhibition, Activity Assay

12) Product Images from "Cyclic Strain Delays the Expression of Tissue Factor Induced by Thrombin in Human Umbilical Vein Endothelial Cells"

Article Title: Cyclic Strain Delays the Expression of Tissue Factor Induced by Thrombin in Human Umbilical Vein Endothelial Cells

Journal: The International Journal of Angiology : Official Publication of the International College of Angiology, Inc

doi: 10.1055/s-0031-1284475

Akt, p38, and ERK activities in HUVEC exposed to CS, Thr, or CS + Thr for 5, 15, and 30 minutes. The activities are indicated by the ratio of phosphorylated/total protein band intensity in western blot. A representative result
Figure Legend Snippet: Akt, p38, and ERK activities in HUVEC exposed to CS, Thr, or CS + Thr for 5, 15, and 30 minutes. The activities are indicated by the ratio of phosphorylated/total protein band intensity in western blot. A representative result

Techniques Used: Western Blot

TF expression in the presence of inhibitors. 10 μM SB203580 was used as a selective inhibitor of p38, 2.5 μM LY294002 as a selective inhibitor of Akt, and 10 μM PD98059 as an inhibitor of ERK. HUVEC were subjected to inhibitors
Figure Legend Snippet: TF expression in the presence of inhibitors. 10 μM SB203580 was used as a selective inhibitor of p38, 2.5 μM LY294002 as a selective inhibitor of Akt, and 10 μM PD98059 as an inhibitor of ERK. HUVEC were subjected to inhibitors

Techniques Used: Expressing

13) Product Images from "Actin-Bundling Protein L-Plastin Regulates T Cell Activation"

Article Title: Actin-Bundling Protein L-Plastin Regulates T Cell Activation

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

doi: 10.4049/jimmunol.1001424

Proximal TCR signaling is normal in LPL −/− CD4 + cells stimulated by immobilized anti-CD3 A), B), D), Immunoblotting. CD4 + cells were mixed with anti-CD3 coated beads at 37°C for indicated time. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with Abs against phospho-tyrosine or GAPDH as loading control (A), phosphorylated forms of ZAP-70, LAT and AKT (B) and total or phospho-ERK (D). C) Calcium flux. Fura 2-AM labeled CD4 + cells were mixed with anti-CD3 coated beads. Time-lapse images were collected at Emission 510nm/Excitation 340 or 380nm, 10 sec/frame, for 10 min. The average ratio of emission 340nm/380nm (coded 0–255) of multiple cells (LPL −/− , n=18; wt, n=22) is plotted over time. E) IL-2 produced by CD4 + cells stimulated with hamster IgG (hIgG)- or anti-CD3- coated beads. Shown are average of duplicates, a representative of two experiments.
Figure Legend Snippet: Proximal TCR signaling is normal in LPL −/− CD4 + cells stimulated by immobilized anti-CD3 A), B), D), Immunoblotting. CD4 + cells were mixed with anti-CD3 coated beads at 37°C for indicated time. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with Abs against phospho-tyrosine or GAPDH as loading control (A), phosphorylated forms of ZAP-70, LAT and AKT (B) and total or phospho-ERK (D). C) Calcium flux. Fura 2-AM labeled CD4 + cells were mixed with anti-CD3 coated beads. Time-lapse images were collected at Emission 510nm/Excitation 340 or 380nm, 10 sec/frame, for 10 min. The average ratio of emission 340nm/380nm (coded 0–255) of multiple cells (LPL −/− , n=18; wt, n=22) is plotted over time. E) IL-2 produced by CD4 + cells stimulated with hamster IgG (hIgG)- or anti-CD3- coated beads. Shown are average of duplicates, a representative of two experiments.

Techniques Used: SDS Page, Labeling, Size-exclusion Chromatography, Produced

14) Product Images from "IL-22 produced by cancer-associated fibroblasts promotes gastric cancer cell invasion via STAT3 and ERK signaling"

Article Title: IL-22 produced by cancer-associated fibroblasts promotes gastric cancer cell invasion via STAT3 and ERK signaling

Journal: British Journal of Cancer

doi: 10.1038/bjc.2014.336

Effect of IL-22 treatment on intracellular signalling in gastric cancer cells. ( A ) Phosphorylation of STAT3, ERK and Akt in AGS and MKN28 cells treated with IL-22. ( B ) Expression of p50 and p60 in AGS and MKN28 cells treated with IL-22. AGS cells (1 × 10 6 ) and MKN28 cells (1 × 10 6 ) were cultured in 6-cm dishes, treated with IL-22 (10 ng ml −1 ) for the indicated time, and extracted protein was analysed using western blotting. ( C ) Effect of anti-IL-22 antibody on IL-22-induced STAT3 and ERK phosphorylation in AGS and MKN28 cells. AGS and MKN28 cells were pretreated with IL-22 antibody (20 μ g ml −1 ) for 45 min and then stimulated with IL-22 (10 ng ml −1 ) for 30 min.
Figure Legend Snippet: Effect of IL-22 treatment on intracellular signalling in gastric cancer cells. ( A ) Phosphorylation of STAT3, ERK and Akt in AGS and MKN28 cells treated with IL-22. ( B ) Expression of p50 and p60 in AGS and MKN28 cells treated with IL-22. AGS cells (1 × 10 6 ) and MKN28 cells (1 × 10 6 ) were cultured in 6-cm dishes, treated with IL-22 (10 ng ml −1 ) for the indicated time, and extracted protein was analysed using western blotting. ( C ) Effect of anti-IL-22 antibody on IL-22-induced STAT3 and ERK phosphorylation in AGS and MKN28 cells. AGS and MKN28 cells were pretreated with IL-22 antibody (20 μ g ml −1 ) for 45 min and then stimulated with IL-22 (10 ng ml −1 ) for 30 min.

Techniques Used: Expressing, Cell Culture, Western Blot

15) Product Images from "Synthetic lethal screening with small molecule inhibitors provides a pathway to rational combination therapies for melanoma"

Article Title: Synthetic lethal screening with small molecule inhibitors provides a pathway to rational combination therapies for melanoma

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-12-0461

The effect of drug combinations on ERK activity Total protein was isolated and immunoblot analyses for pERK, tERK, and tubulin were performed from cells treated as follows: (A) DM331 were incubated with vehicle, 2.5 μM sorafenib, 5 μM sorafenib, 50 μM diclofenac, or sorafenib and diclofenac for 1 h. (B) Quantification of Western blot analysis (n≥4). (C) DM331 cells incubated with vehicle, 1 nM PD325901, 5 nM PD325901, 50 μM diclofenac, or PD325901 and diclofenac for 1 h. (D) Quantification of Western blot analysis (n=3).
Figure Legend Snippet: The effect of drug combinations on ERK activity Total protein was isolated and immunoblot analyses for pERK, tERK, and tubulin were performed from cells treated as follows: (A) DM331 were incubated with vehicle, 2.5 μM sorafenib, 5 μM sorafenib, 50 μM diclofenac, or sorafenib and diclofenac for 1 h. (B) Quantification of Western blot analysis (n≥4). (C) DM331 cells incubated with vehicle, 1 nM PD325901, 5 nM PD325901, 50 μM diclofenac, or PD325901 and diclofenac for 1 h. (D) Quantification of Western blot analysis (n=3).

Techniques Used: Activity Assay, Isolation, Incubation, Western Blot

16) Product Images from "Oncogenic B-RAF Negatively Regulates the Tumor Suppressor LKB1 to Promote Melanoma Cell Proliferation"

Article Title: Oncogenic B-RAF Negatively Regulates the Tumor Suppressor LKB1 to Promote Melanoma Cell Proliferation

Journal: Molecular cell

doi: 10.1016/j.molcel.2008.12.026

Phosphorylation of Ser325 and Ser428 of LKB1 by two downstream kinases of B-RAF, ERK and p90Rsk, respectively.
Figure Legend Snippet: Phosphorylation of Ser325 and Ser428 of LKB1 by two downstream kinases of B-RAF, ERK and p90Rsk, respectively.

Techniques Used:

17) Product Images from "Identification of SHCBP1 as a novel downstream target gene of SS18-SSX1 and its functional analysis in progression of synovial sarcoma"

Article Title: Identification of SHCBP1 as a novel downstream target gene of SS18-SSX1 and its functional analysis in progression of synovial sarcoma

Journal: Oncotarget

doi: 10.18632/oncotarget.11651

SHCBP1 exerts anti-apoptotic effect via activating MAPK/ERK and PI3K/AKT/mTOR signaling pathways and enhancing the expression of cyclin D1 A and B. Apoptosis was determined by flow cytometry in SHCBP1-overexpressing ( A ) and SHCBP1-silenced cells ( B ). Cells stained with annexin-V were considered as apoptosis. The apoptotic index was defined as the percentage of apoptotic cells. ( C ) The levels of phosphorylated MEK, total MEK, phosphorylated ERK, total ERK, phosphorylated Akt, total Akt, and cyclin D1 were detected in control and SHCBP1-siRNA cells by western blot analysis. β-actin was used as the loading control. Values are mean ± SD of 3 independent experiments; ** p
Figure Legend Snippet: SHCBP1 exerts anti-apoptotic effect via activating MAPK/ERK and PI3K/AKT/mTOR signaling pathways and enhancing the expression of cyclin D1 A and B. Apoptosis was determined by flow cytometry in SHCBP1-overexpressing ( A ) and SHCBP1-silenced cells ( B ). Cells stained with annexin-V were considered as apoptosis. The apoptotic index was defined as the percentage of apoptotic cells. ( C ) The levels of phosphorylated MEK, total MEK, phosphorylated ERK, total ERK, phosphorylated Akt, total Akt, and cyclin D1 were detected in control and SHCBP1-siRNA cells by western blot analysis. β-actin was used as the loading control. Values are mean ± SD of 3 independent experiments; ** p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Western Blot

18) Product Images from "Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38? Activation"

Article Title: Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38? Activation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.16.6957-6966.2004

Anergic CD4 + T cells show decreased activation of ERK and JNK and increased activation of p38 MAPK in response to TCR stimulation. (A) CD4 + T cells were purified from untreated Vβ8.1-tg mice (naive) or Mls-1 a -treated Vβ8.1-tg mice (anergic) by treatment of lymph node cells with anti-CD8 MAb and complement, followed by nylon wool column enrichment. Cells were cultured for 3 h at 37°C in complete medium to decrease basal levels of MAPK activity. Cells then were placed in anti-TCR MAb (10 μg/ml)-coated plates for various times and lysed, and the levels of phosphorylated and total MAPKs were determined by immunoblotting with specific antibodies. Representative results from three independent experiments are shown. (B) In vitro immune complex kinase assay of p38α and ERK1. Naive or anergic CD4 + T cells were prepared as described for panel A and placed in anti-TCR MAb (10 μg/ml)-coated plates in the presence or absence of SB203580 for various times. Cell lysates were immunoprecipitated with anti-p38α or anti-ERK1 antibody, and in vitro kinase assays of the immunoprecipitates were performed with ATF2 and maltose-binding protein (MBP) as substrates for p38α and ERK1, respectively. Proteins were separated by SDS-PAGE and analyzed by autoradiography. The quantities of p38α and ERK1 were assessed by probing the immunoblots with antibodies specific for p38α or ERK1. Representative results from three independent experiments are shown.
Figure Legend Snippet: Anergic CD4 + T cells show decreased activation of ERK and JNK and increased activation of p38 MAPK in response to TCR stimulation. (A) CD4 + T cells were purified from untreated Vβ8.1-tg mice (naive) or Mls-1 a -treated Vβ8.1-tg mice (anergic) by treatment of lymph node cells with anti-CD8 MAb and complement, followed by nylon wool column enrichment. Cells were cultured for 3 h at 37°C in complete medium to decrease basal levels of MAPK activity. Cells then were placed in anti-TCR MAb (10 μg/ml)-coated plates for various times and lysed, and the levels of phosphorylated and total MAPKs were determined by immunoblotting with specific antibodies. Representative results from three independent experiments are shown. (B) In vitro immune complex kinase assay of p38α and ERK1. Naive or anergic CD4 + T cells were prepared as described for panel A and placed in anti-TCR MAb (10 μg/ml)-coated plates in the presence or absence of SB203580 for various times. Cell lysates were immunoprecipitated with anti-p38α or anti-ERK1 antibody, and in vitro kinase assays of the immunoprecipitates were performed with ATF2 and maltose-binding protein (MBP) as substrates for p38α and ERK1, respectively. Proteins were separated by SDS-PAGE and analyzed by autoradiography. The quantities of p38α and ERK1 were assessed by probing the immunoblots with antibodies specific for p38α or ERK1. Representative results from three independent experiments are shown.

Techniques Used: Activation Assay, Purification, Mouse Assay, Cell Culture, Activity Assay, In Vitro, Immune Complex Kinase Assay, Immunoprecipitation, Binding Assay, SDS Page, Autoradiography, Western Blot

The inhibition of ERK kinase activity by TAB1 is dependent on p38. (A) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured with various doses of PMA (0 to 60 ng/ml) for 10 min, and the levels of phosphorylated ERK and total ERK were determined by immunoblotting with specific antibodies. (B) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured in the presence (SB) or absence (Cont) of SB203580 (10 μM) for 2 h and subsequently stimulated with PMA (0, 10, or 20 ng/ml) for 10 min. Cells were lysed, and the levels of phosphorylated ERK and total ERK were determined by immunoblotting with specific antibodies. (C) Mock-2B4 cells were transduced with a retroviral supernatant containing empty vector (Mock/Mock) or p38DN (Mock/p38DN) to establish stable cell lines. TAB1-2B4 cells were also transduced to establish cell lines expressing empty vector (TAB1/Mock) or p38DN cDNA (TAB1/p38DN). These cell lines were stimulated with PMA (0, 5, or 25 ng/ml) for 10 min and lysed, and the levels of phosphorylated MAPks and total MAPKs were determined by immunoblotting with specific antibodies. Representative results from two independent experiments are shown. (D) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were treated or not treated with SB203580 (10 μM) for 2 h and stimulated with PMA (20 ng/ml) and ionomycin (Iono; 2 μM) for 8 h in the presence (SB) or absence (Cont) of SB203580, respectively. Cells were subjected to RT-PCR analysis for IL-2 and G3PDH mRNAs. Representative results from two independent experiments are shown. (E) Mock/Mock, Mock/p38DN, TAB1/Mock, and TAB1/p38DN cells were stimulated with PMA (0, 5, or 25 ng/ml) and ionomycin (2 μM) for 8 h. Cells were subjected to RT-PCR analysis for IL-2, IL-10, and G3PDH mRNAs. Representative results from two independent experiments are shown. (F) Mock/Mock, Mock/p38DN, TAB1/Mock, and TAB1/p38DN cells were stimulated with anti-TCR MAb (H57; 10 μg/ml) for 24 h. The levels of IL-2 in culture supernatants were determined by an ELISA. IL-2 was not detectable in cultures without anti-TCR MAb.
Figure Legend Snippet: The inhibition of ERK kinase activity by TAB1 is dependent on p38. (A) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured with various doses of PMA (0 to 60 ng/ml) for 10 min, and the levels of phosphorylated ERK and total ERK were determined by immunoblotting with specific antibodies. (B) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were cultured in the presence (SB) or absence (Cont) of SB203580 (10 μM) for 2 h and subsequently stimulated with PMA (0, 10, or 20 ng/ml) for 10 min. Cells were lysed, and the levels of phosphorylated ERK and total ERK were determined by immunoblotting with specific antibodies. (C) Mock-2B4 cells were transduced with a retroviral supernatant containing empty vector (Mock/Mock) or p38DN (Mock/p38DN) to establish stable cell lines. TAB1-2B4 cells were also transduced to establish cell lines expressing empty vector (TAB1/Mock) or p38DN cDNA (TAB1/p38DN). These cell lines were stimulated with PMA (0, 5, or 25 ng/ml) for 10 min and lysed, and the levels of phosphorylated MAPks and total MAPKs were determined by immunoblotting with specific antibodies. Representative results from two independent experiments are shown. (D) Mock-2B4 cells (Mock) and TAB1-2B4 cells (TAB1) were treated or not treated with SB203580 (10 μM) for 2 h and stimulated with PMA (20 ng/ml) and ionomycin (Iono; 2 μM) for 8 h in the presence (SB) or absence (Cont) of SB203580, respectively. Cells were subjected to RT-PCR analysis for IL-2 and G3PDH mRNAs. Representative results from two independent experiments are shown. (E) Mock/Mock, Mock/p38DN, TAB1/Mock, and TAB1/p38DN cells were stimulated with PMA (0, 5, or 25 ng/ml) and ionomycin (2 μM) for 8 h. Cells were subjected to RT-PCR analysis for IL-2, IL-10, and G3PDH mRNAs. Representative results from two independent experiments are shown. (F) Mock/Mock, Mock/p38DN, TAB1/Mock, and TAB1/p38DN cells were stimulated with anti-TCR MAb (H57; 10 μg/ml) for 24 h. The levels of IL-2 in culture supernatants were determined by an ELISA. IL-2 was not detectable in cultures without anti-TCR MAb.

Techniques Used: Inhibition, Activity Assay, Cell Culture, Transduction, Plasmid Preparation, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

19) Product Images from "TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii"

Article Title: TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

Journal: Infection and Immunity

doi: 10.1128/IAI.72.10.5662-5667.2004

TRAF6 is essential for p38 and ERK phosphorylation in response to toxoplasma antigen. WT and TRAF6 −/− macrophages derived from splenic precursors were primed with IFN-γ for 24 h and stimulated with either LPS or STAg for 45 min. Cell lysates were analyzed by Western blotting for the presence of total and phosphorylated forms of p38 (A) or total and phosphorylated forms of ERK1 and ERK2 (B). KO, knockout mutant.
Figure Legend Snippet: TRAF6 is essential for p38 and ERK phosphorylation in response to toxoplasma antigen. WT and TRAF6 −/− macrophages derived from splenic precursors were primed with IFN-γ for 24 h and stimulated with either LPS or STAg for 45 min. Cell lysates were analyzed by Western blotting for the presence of total and phosphorylated forms of p38 (A) or total and phosphorylated forms of ERK1 and ERK2 (B). KO, knockout mutant.

Techniques Used: Derivative Assay, Western Blot, Knock-Out, Mutagenesis

Activation of ERK inhibits IL-12 production in response to toxoplasma antigen. (A) BMMφ were primed with IFN-γ for 24 h and then treated with STAg (50 μg/ml). Cell lysates were analyzed for phosphorylated and total levels of ERK1 and ERK2 at the times indicated. (B) BMMφ were primed with IFN-γ for 24 h and then treated with SB202474 (10 μM) (negative control) or PD98059 (10 μM) for 1 h before being stimulated with STAg (50 μg/ml) for 20 h. Culture supernatants were then analyzed by ELISA for IL-12(p40) production. The data indicate mean values ± SD of results from triplicate wells. Each experiment was performed three times.
Figure Legend Snippet: Activation of ERK inhibits IL-12 production in response to toxoplasma antigen. (A) BMMφ were primed with IFN-γ for 24 h and then treated with STAg (50 μg/ml). Cell lysates were analyzed for phosphorylated and total levels of ERK1 and ERK2 at the times indicated. (B) BMMφ were primed with IFN-γ for 24 h and then treated with SB202474 (10 μM) (negative control) or PD98059 (10 μM) for 1 h before being stimulated with STAg (50 μg/ml) for 20 h. Culture supernatants were then analyzed by ELISA for IL-12(p40) production. The data indicate mean values ± SD of results from triplicate wells. Each experiment was performed three times.

Techniques Used: Activation Assay, Negative Control, Enzyme-linked Immunosorbent Assay

20) Product Images from "Cell Cycle Phase Regulates Glucocorticoid Receptor Function"

Article Title: Cell Cycle Phase Regulates Glucocorticoid Receptor Function

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022289

GR regulation of kinases is dependent on cell cycle phase. A549 (A–C) or HeLa (D–F) cells were treated with vehicle or nocodazole for 16 hours, washed and released into mitosis in the presence of 100 nM dex for up to 60 minutes. Cells were lysed and immunoblotted for phospho-PKB and PKB (A), or phospho-ERK and ERK (D). Immunolabelling was quantified by densitometry using ImageJ, where both cell cycle effects (B, E) and Gc-dependent effects (C, F) on kinase activity are depicted. * indicates p
Figure Legend Snippet: GR regulation of kinases is dependent on cell cycle phase. A549 (A–C) or HeLa (D–F) cells were treated with vehicle or nocodazole for 16 hours, washed and released into mitosis in the presence of 100 nM dex for up to 60 minutes. Cells were lysed and immunoblotted for phospho-PKB and PKB (A), or phospho-ERK and ERK (D). Immunolabelling was quantified by densitometry using ImageJ, where both cell cycle effects (B, E) and Gc-dependent effects (C, F) on kinase activity are depicted. * indicates p

Techniques Used: Activity Assay

21) Product Images from "Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation"

Article Title: Transforming Growth Factor ? Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021170

TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.
Figure Legend Snippet: TGF-β does not inhibit Lck or ZAP-70 phosphorylations but inhibits ERK phosphorylation. Whole cell lysates were prepared from CD4 + T cells stimulated by cross-linking anti-CD3 and anti-CD28 bound to cells under neutral conditions (IL-2, 50 U/ml) in the presence or absence of TGF-β (50 pM). Total cell lysates were prepared and analyzed for the phosphorylation of (A) Lck and ZAP-70 or (B) ERK, JNK, and GSK-3.

Techniques Used:

22) Product Images from "Chemical Constituents and an Antineuroinflammatory Lignan, Savinin from the Roots of Acanthopanax henryi"

Article Title: Chemical Constituents and an Antineuroinflammatory Lignan, Savinin from the Roots of Acanthopanax henryi

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2019/1856294

Effects of compound 3 on the LPS-induced activation of MAPK pathways in BV2 microglial cells. Lysates were prepared from cells pretreated with/without the indicated concentrations of compound 3 for 3 h and then with LPS (1 μ g/mL) for 30 min. The levels of p-p38, p38, p-ERK, ERK, p-JNK, and JNK were determined by Western blot analysis. Actin was used as a loading control. The experiment was repeated three times, and similar results were obtained.
Figure Legend Snippet: Effects of compound 3 on the LPS-induced activation of MAPK pathways in BV2 microglial cells. Lysates were prepared from cells pretreated with/without the indicated concentrations of compound 3 for 3 h and then with LPS (1 μ g/mL) for 30 min. The levels of p-p38, p38, p-ERK, ERK, p-JNK, and JNK were determined by Western blot analysis. Actin was used as a loading control. The experiment was repeated three times, and similar results were obtained.

Techniques Used: Activation Assay, Western Blot

23) Product Images from "Bimodal Effect on Pancreatic ?-Cells of Secretory Products From Normal or Insulin-Resistant Human Skeletal Muscle"

Article Title: Bimodal Effect on Pancreatic ?-Cells of Secretory Products From Normal or Insulin-Resistant Human Skeletal Muscle

Journal: Diabetes

doi: 10.2337/db10-1178

Action of conditioned medium on Akt, ERK, AS160, and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P
Figure Legend Snippet: Action of conditioned medium on Akt, ERK, AS160, and Akt substrates phosphorylation after glucose stimulation, or IRS-1 and -2 protein expression in rat primary β-cells. Conditioned media and abbreviations as described in the legend to Fig. 3 . After 24-h culture in conditioned medium, β-cells were incubated 1 h at 2.8 mmol/L or 16.7 mmol/L glucose (Glc). Open bars = 2.8 mmol/L glucose; closed bars = 16.7 mmol/L glucose. Western blots were scanned and data normalized to total protein or actin as indicated. N = 3 independent experiments. A : Akt Ser 473 phosphorylation. * P

Techniques Used: Expressing, Incubation, Gas Chromatography, Western Blot

24) Product Images from "Hwangryunhaedok-Tang Exerts Neuropreventive Effect on Memory Impairment by Reducing Cholinergic System Dysfunction and Inflammatory Response in a Vascular Dementia Rat Model"

Article Title: Hwangryunhaedok-Tang Exerts Neuropreventive Effect on Memory Impairment by Reducing Cholinergic System Dysfunction and Inflammatory Response in a Vascular Dementia Rat Model

Journal: Molecules

doi: 10.3390/molecules24020343

Effect of HRT on inflammatory responses in the BCCAO-induced VaD rats. ( A ) Immunohistological analysis was carried out to examine the expression of ionized calcium binding adaptor molecule 1 (Iba-1), a microglial marker, in the hippocampus of BCCAO rats. Representative photomicrographs are shown at magnifications of 400×. The immunohistochemical signal intensity was calculated in a brain tissue section from each rat using Image J software. The staining intensity results are reported in arbitrary units (AU). ( B ) Hippocampal tissues were lysed and subjected to Western blotting for detecting phosphorylated mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Expression levels were normalized to the expression of actin. Data are presented as means ± SEM ( n = 8); ** p
Figure Legend Snippet: Effect of HRT on inflammatory responses in the BCCAO-induced VaD rats. ( A ) Immunohistological analysis was carried out to examine the expression of ionized calcium binding adaptor molecule 1 (Iba-1), a microglial marker, in the hippocampus of BCCAO rats. Representative photomicrographs are shown at magnifications of 400×. The immunohistochemical signal intensity was calculated in a brain tissue section from each rat using Image J software. The staining intensity results are reported in arbitrary units (AU). ( B ) Hippocampal tissues were lysed and subjected to Western blotting for detecting phosphorylated mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Expression levels were normalized to the expression of actin. Data are presented as means ± SEM ( n = 8); ** p

Techniques Used: Expressing, Binding Assay, Marker, Immunohistochemistry, Software, Staining, Western Blot

25) Product Images from "Pretreatment of ferulic acid attenuates inflammation and oxidative stress in a rat model of lipopolysaccharide-induced acute respiratory distress syndrome"

Article Title: Pretreatment of ferulic acid attenuates inflammation and oxidative stress in a rat model of lipopolysaccharide-induced acute respiratory distress syndrome

Journal: International Journal of Immunopathology and Pharmacology

doi: 10.1177/0394632017750518

FA down-regulates mitogen-activated protein kinase (MAPK) signaling pathways in rats with LPS administration. (a and b) MAPK signaling pathway-related proteins (phosphorylated and total proteins of p38, ERK and JNK) were analyzed by immunoblotting. GAPDH was employed as the loading control. N = 8. Data are presented as mean ± SD. * P
Figure Legend Snippet: FA down-regulates mitogen-activated protein kinase (MAPK) signaling pathways in rats with LPS administration. (a and b) MAPK signaling pathway-related proteins (phosphorylated and total proteins of p38, ERK and JNK) were analyzed by immunoblotting. GAPDH was employed as the loading control. N = 8. Data are presented as mean ± SD. * P

Techniques Used:

26) Product Images from "Sulfuretin Attenuates MPP+-Induced Neurotoxicity through Akt/GSK3β and ERK Signaling Pathways"

Article Title: Sulfuretin Attenuates MPP+-Induced Neurotoxicity through Akt/GSK3β and ERK Signaling Pathways

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18122753

LY294002 suppresses sulfuretin-induced protection against MPP + , whereas SB415286 reverses MPP + -induced cytotoxicity. SH-SY5Y cells were pretreated with or without LY294002 (10 μM) for 2 h, followed by treatment with or without sulfuretin (40 μM) for 2 h and exposed to MPP + (1 mM) for 24 h. ( A ) Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). ( B ) Protein levels of p-Akt, Akt, p-GSK3β, GSK3β, p-ERK, ERK, and GAPDH were determined by Western blot analysis. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). ( C ) SH-SY5Y cells were pretreated with or without SB415286 (20 μM) for 2 h, and then exposed to MPP + (1 mM) for 24 h. Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). Differences are statistically significant at ** p
Figure Legend Snippet: LY294002 suppresses sulfuretin-induced protection against MPP + , whereas SB415286 reverses MPP + -induced cytotoxicity. SH-SY5Y cells were pretreated with or without LY294002 (10 μM) for 2 h, followed by treatment with or without sulfuretin (40 μM) for 2 h and exposed to MPP + (1 mM) for 24 h. ( A ) Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). ( B ) Protein levels of p-Akt, Akt, p-GSK3β, GSK3β, p-ERK, ERK, and GAPDH were determined by Western blot analysis. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). ( C ) SH-SY5Y cells were pretreated with or without SB415286 (20 μM) for 2 h, and then exposed to MPP + (1 mM) for 24 h. Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). Differences are statistically significant at ** p

Techniques Used: MTT Assay, Western Blot

MPP + decreases Akt/GSK3β and ERK phosphorylation and increases p53 expression, whereas sulfuretin reverses its effect. SH-SY5Y cells were pretreated with sulfuretin for 2 h and then treated with MPP + for 24 h. After cell lysis, the extracted proteins were subjected to Western blot analysis using specific antibodies. Protein levels of ( A ) p-Akt, Akt, p-GSK3β, GSK3β, p-CREB, and GAPDH; ( B ) p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). Differences are statistically significant at * p
Figure Legend Snippet: MPP + decreases Akt/GSK3β and ERK phosphorylation and increases p53 expression, whereas sulfuretin reverses its effect. SH-SY5Y cells were pretreated with sulfuretin for 2 h and then treated with MPP + for 24 h. After cell lysis, the extracted proteins were subjected to Western blot analysis using specific antibodies. Protein levels of ( A ) p-Akt, Akt, p-GSK3β, GSK3β, p-CREB, and GAPDH; ( B ) p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). Differences are statistically significant at * p

Techniques Used: Expressing, Lysis, Western Blot

PD98059 suppresses sulfuretin-induced protection against MPP + . SH-SY5Y cells were pretreated with or without PD98059 (10 μM) for 2 h, followed by treatment with or without sulfuretin (40 μM) for 2 h, and exposed to MPP + (1 mM) for 24 h. ( A ) Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). ( B ) Protein levels of p-ERK, ERK, p-Akt, Akt, p-GSK3β, GSK3β, and GAPDH were determined by Western blot analysis. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). Differences are statistically significant at * p
Figure Legend Snippet: PD98059 suppresses sulfuretin-induced protection against MPP + . SH-SY5Y cells were pretreated with or without PD98059 (10 μM) for 2 h, followed by treatment with or without sulfuretin (40 μM) for 2 h, and exposed to MPP + (1 mM) for 24 h. ( A ) Cell viability was measured by MTT assay. Values are presented relative to control as mean percentage change ± S.D. ( n = 3). ( B ) Protein levels of p-ERK, ERK, p-Akt, Akt, p-GSK3β, GSK3β, and GAPDH were determined by Western blot analysis. Representative blots and their densitometric quantification are shown. Values are presented relative to control as mean fold change ± S.D. ( n = 3). Differences are statistically significant at * p

Techniques Used: MTT Assay, Western Blot

27) Product Images from "The oncoprotein gankyrin promotes the development of colitis-associated cancer through activation of STAT3"

Article Title: The oncoprotein gankyrin promotes the development of colitis-associated cancer through activation of STAT3

Journal: Oncotarget

doi: 10.18632/oncotarget.14983

Loss of gankyrin in myeloid cells results in reduced expression of cytokines and stem cell markers A . RNA was extracted from non-tumor colon of tumor-harboring Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice. Relative amounts of mRNA were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 5). B . Effect of gankyrin on gene expression in colonic lamina propria (LP) cells. LP cells were isolated from DSS-treated Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice and cytokine mRNA expression was analyzed by real-time qPCR. The mRNA expression levels in LP cells from non-treated WT mice were set as 1. C . RNA was extracted from tumor tissues of Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice. Relative amounts of mRNA of ERK target genes cMyc, and stem cell markers Sox9, Bmi1, Lgr5 and Ascl2 were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 5). D . Relative amounts of mRNA of TNF-α and Pim1 from THP-1 cells transfected with gankyrin RNAi or control RNAi were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 3).
Figure Legend Snippet: Loss of gankyrin in myeloid cells results in reduced expression of cytokines and stem cell markers A . RNA was extracted from non-tumor colon of tumor-harboring Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice. Relative amounts of mRNA were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 5). B . Effect of gankyrin on gene expression in colonic lamina propria (LP) cells. LP cells were isolated from DSS-treated Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice and cytokine mRNA expression was analyzed by real-time qPCR. The mRNA expression levels in LP cells from non-treated WT mice were set as 1. C . RNA was extracted from tumor tissues of Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice. Relative amounts of mRNA of ERK target genes cMyc, and stem cell markers Sox9, Bmi1, Lgr5 and Ascl2 were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 5). D . Relative amounts of mRNA of TNF-α and Pim1 from THP-1 cells transfected with gankyrin RNAi or control RNAi were determined by real-time qPCR and normalized to the amount of actin mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value of 1.0. Data are means ± SEM (n = 3).

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Transfection

Gankyrin activates STAT3 in non-tumor tissues and ERK in tumors A . Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice were challenged with AOM and DSS. Homogenates of non-treated colons (Control), non-tumor colon tissues (Non-tumor) and tumors (Tumor) were gel-separated and immunoblotted with the indicated antibodies. B . Relative kinase activities in non-tumor colon tissues (Non-tumor) and tumors (Tumor) isolated from Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice challenged with AOM and DSS were assessed by densitometry. Kinase activity in untreated Gankyrin f/f mice was given an arbitrary value of 1. Data are means ± SEM. C, D . Representative images of immunohistochemical detection of phosphorylated STAT3 (C) and phosphorylated ERK (D) in non-tumor colon tissues (Non-tumor) and tumors (Tumor). Scale bar, 100 μm.
Figure Legend Snippet: Gankyrin activates STAT3 in non-tumor tissues and ERK in tumors A . Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice were challenged with AOM and DSS. Homogenates of non-treated colons (Control), non-tumor colon tissues (Non-tumor) and tumors (Tumor) were gel-separated and immunoblotted with the indicated antibodies. B . Relative kinase activities in non-tumor colon tissues (Non-tumor) and tumors (Tumor) isolated from Mx1-Cre;Gankyrin f/f (Mx1-Cre;GK f/f ) mice and Gankyrin f/f (GK f/f ) mice challenged with AOM and DSS were assessed by densitometry. Kinase activity in untreated Gankyrin f/f mice was given an arbitrary value of 1. Data are means ± SEM. C, D . Representative images of immunohistochemical detection of phosphorylated STAT3 (C) and phosphorylated ERK (D) in non-tumor colon tissues (Non-tumor) and tumors (Tumor). Scale bar, 100 μm.

Techniques Used: Mouse Assay, Isolation, Activity Assay, Immunohistochemistry

Chronic inflammation increases gankyrin expression in the colonic mucosa of patients with IBD In inflammatory cells, gankyrin activates STAT3 by binding to SHP-1, leading to enhanced inflammation. These augmented inflammatory responses would activate ERK in tumors and eventually promote colitis-associated cancer.
Figure Legend Snippet: Chronic inflammation increases gankyrin expression in the colonic mucosa of patients with IBD In inflammatory cells, gankyrin activates STAT3 by binding to SHP-1, leading to enhanced inflammation. These augmented inflammatory responses would activate ERK in tumors and eventually promote colitis-associated cancer.

Techniques Used: Expressing, Binding Assay

28) Product Images from "Human regulatory T cells induce T-lymphocyte senescence"

Article Title: Human regulatory T cells induce T-lymphocyte senescence

Journal: Blood

doi: 10.1182/blood-2012-03-416040

Cell-cycle–regulatory molecules and phosphorylated activation of MAPKs are involved in Treg-induced senescence. (A) Cell-cycle–regulatory molecules p21, p16, and p53 are involved in human Treg-induced T-cell senescence. (B) CD4 + CD25 hi FoxP3 + Treg cells induced phosphorylation of ERK and p38 in senescent CD4 + T cells. Results from Western blotting analyses of phosphorylated activation of MAPKs are shown in the left panel. Phosphorylated ERK and p38 protein levels shown in the right histogram were analyzed quantitatively and compared against the β-actin expression level with a densitometer. Results shown in the histogram are means ± SD from 3 independent experiments. * P
Figure Legend Snippet: Cell-cycle–regulatory molecules and phosphorylated activation of MAPKs are involved in Treg-induced senescence. (A) Cell-cycle–regulatory molecules p21, p16, and p53 are involved in human Treg-induced T-cell senescence. (B) CD4 + CD25 hi FoxP3 + Treg cells induced phosphorylation of ERK and p38 in senescent CD4 + T cells. Results from Western blotting analyses of phosphorylated activation of MAPKs are shown in the left panel. Phosphorylated ERK and p38 protein levels shown in the right histogram were analyzed quantitatively and compared against the β-actin expression level with a densitometer. Results shown in the histogram are means ± SD from 3 independent experiments. * P

Techniques Used: Activation Assay, Western Blot, Expressing

29) Product Images from "Modulation of NMDAR Subunit Expression by TRPM2 Channels Regulates Neuronal Vulnerability to Ischemic Cell Death"

Article Title: Modulation of NMDAR Subunit Expression by TRPM2 Channels Regulates Neuronal Vulnerability to Ischemic Cell Death

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1729-13.2013

TRPM2 −/− increases activity in ERK and Akt pathways. Western blots of whole hippocampi from WT and TRPM2 −/− animals detecting changes in expression of proteins responsible for downstream NMDAR-mediated cell survival mechanisms. A , Basal levels of Akt had no change in expression between WT and TRPM2 −/− . B , C , However, phospho-Akt was increased at both phosphorylation sites (49.39 ± 8.26% in Ser-473; 65.11 ± 13.78% in Thr-308; n = 4; * p
Figure Legend Snippet: TRPM2 −/− increases activity in ERK and Akt pathways. Western blots of whole hippocampi from WT and TRPM2 −/− animals detecting changes in expression of proteins responsible for downstream NMDAR-mediated cell survival mechanisms. A , Basal levels of Akt had no change in expression between WT and TRPM2 −/− . B , C , However, phospho-Akt was increased at both phosphorylation sites (49.39 ± 8.26% in Ser-473; 65.11 ± 13.78% in Thr-308; n = 4; * p

Techniques Used: Activity Assay, Western Blot, Expressing

30) Product Images from "Cell Type-Specific Interleukin-1β Signaling in the CNS"

Article Title: Cell Type-Specific Interleukin-1β Signaling in the CNS

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.5712-03.2004

Western blot analysis for phosphorylation of CREB and the different MAP kinases in hippocampal neurons. Cultured hippocampal neurons were treated with IL-1β (10 ng/ml) for the indicated times, lysed, and analyzed by Western blot for P-CREB ( A ), P-JNK ( B ), P-ERK ( C ), or P-p38 ( D ). Blots were stripped and reprobed for total CREB ( A ), JNK ( B ), and p38 ( D ) or ERK (data not shown because no differences in P-ERK were seen).
Figure Legend Snippet: Western blot analysis for phosphorylation of CREB and the different MAP kinases in hippocampal neurons. Cultured hippocampal neurons were treated with IL-1β (10 ng/ml) for the indicated times, lysed, and analyzed by Western blot for P-CREB ( A ), P-JNK ( B ), P-ERK ( C ), or P-p38 ( D ). Blots were stripped and reprobed for total CREB ( A ), JNK ( B ), and p38 ( D ) or ERK (data not shown because no differences in P-ERK were seen).

Techniques Used: Western Blot, Cell Culture

31) Product Images from "Med1 plays a critical role in the development of tamoxifen resistance"

Article Title: Med1 plays a critical role in the development of tamoxifen resistance

Journal: Carcinogenesis

doi: 10.1093/carcin/bgs105

Increased expression and phosphorylation of Med1 in tamoxifen-resistant cells due to persistent activation of ERK. ( A ) Immunoblot analysis of Med1, ERK, phospho-Med1 and phospho-ERK in MCF7-P and MCF7-OHT. ( B ) Immunoblot analysis of pMed1, pERK and ERK
Figure Legend Snippet: Increased expression and phosphorylation of Med1 in tamoxifen-resistant cells due to persistent activation of ERK. ( A ) Immunoblot analysis of Med1, ERK, phospho-Med1 and phospho-ERK in MCF7-P and MCF7-OHT. ( B ) Immunoblot analysis of pMed1, pERK and ERK

Techniques Used: Expressing, Activation Assay

32) Product Images from "Sprouty2 enhances the tumorigenic potential of glioblastoma cells"

Article Title: Sprouty2 enhances the tumorigenic potential of glioblastoma cells

Journal: Neuro-Oncology

doi: 10.1093/neuonc/noy028

Silencing of SPRY2 increases ERK and Akt activation, and MEK inhibitor prevents the decreased cell proliferation of SPRY2 KD. (A) U87 or GSC1 cells transduced with tet-Ctrlsh or tet-SPRY2sh were treated with or without 1 µg/mL doxycycline for 72 hours, serum- (U87) or EGF and FGF2- (GSC1) starved overnight, then treated with EGF (50 ng/mL) for the indicated times. Immunoblotting was performed with the indicated antibodies. (B) Immunoblots for the indicated proteins in U87 tet-SPRY2sh cells (± Doxy) pretreated with the indicated concentrations of PD98059 or dimethyl sulfoxide (DMSO), and stimulated with EGF (20 ng/mL) for 20 minutes. (C) Measurement of cell proliferation of U87 tet-SPRY2sh cells (± Doxy) in the presence of DMSO or 10 µM PD98059. Mean ± SEM of at least 3 independent experiments. ** P
Figure Legend Snippet: Silencing of SPRY2 increases ERK and Akt activation, and MEK inhibitor prevents the decreased cell proliferation of SPRY2 KD. (A) U87 or GSC1 cells transduced with tet-Ctrlsh or tet-SPRY2sh were treated with or without 1 µg/mL doxycycline for 72 hours, serum- (U87) or EGF and FGF2- (GSC1) starved overnight, then treated with EGF (50 ng/mL) for the indicated times. Immunoblotting was performed with the indicated antibodies. (B) Immunoblots for the indicated proteins in U87 tet-SPRY2sh cells (± Doxy) pretreated with the indicated concentrations of PD98059 or dimethyl sulfoxide (DMSO), and stimulated with EGF (20 ng/mL) for 20 minutes. (C) Measurement of cell proliferation of U87 tet-SPRY2sh cells (± Doxy) in the presence of DMSO or 10 µM PD98059. Mean ± SEM of at least 3 independent experiments. ** P

Techniques Used: Activation Assay, Transduction, Western Blot

33) Product Images from "VEGFR2 regulates endothelial differentiation of colon cancer cells"

Article Title: VEGFR2 regulates endothelial differentiation of colon cancer cells

Journal: BMC Cancer

doi: 10.1186/s12885-017-3578-9

SKLB1002 inhibited tube-like structures formation and VE-cadherin expression of HCT116 cells. a The tube-like structures formed by HCT116 cells with or without SKLB1002 treatment (left). Red arrows indicate the typical tube-like structures. Scale bar: 100 μm; Quantitative analysis of the mean number of tube-like structures and shown as mean ± SD (right). b Protein expressions of VEGFR2, p-VEGFR2, FAK, p-FAK, ERK, p-ERK and VE-cadherin in HCT116 cells with or without SKLB1002 treatment were determined by western blotting analysis. Representative data of three experiments are shown
Figure Legend Snippet: SKLB1002 inhibited tube-like structures formation and VE-cadherin expression of HCT116 cells. a The tube-like structures formed by HCT116 cells with or without SKLB1002 treatment (left). Red arrows indicate the typical tube-like structures. Scale bar: 100 μm; Quantitative analysis of the mean number of tube-like structures and shown as mean ± SD (right). b Protein expressions of VEGFR2, p-VEGFR2, FAK, p-FAK, ERK, p-ERK and VE-cadherin in HCT116 cells with or without SKLB1002 treatment were determined by western blotting analysis. Representative data of three experiments are shown

Techniques Used: Expressing, Western Blot

34) Product Images from "Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer"

Article Title: Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.25435

Periostin promotes Ex3LL-cell proliferation and intracellular signaling in vitro (A) GFP fluorescence in Ex3LL-GFP cells co-cultured with periostin −/− or periostin +/+ fibroblasts ( n =5). Values on the Y-axis are normalized to the value on day 0. (B) Effect of periostin stimulation on Ex3LL proliferation, assayed by MTT ( n =5). Values on the Y-axis are normalized to the value on day 0. (C) Ex3LL cells were incubated for 24 h with medium containing 0.1% FBS and then treated with 1,000 ng/ml periostin for 5, 10, 30, or 60 min. The presence of ERK, AKT, FAK, and NF-κB and their phosphorylated forms [pERK (Thr202/Tyr204), pAKT (Thr308), pFAK (Tyr925), and p-NF-kB (Ser536)] in the Ex3LL cells was assessed by western blotting. Ratios of pERK to total ERK, based on band density analyzed with ImageJ software, are shown. (D) Ex3LL cell proliferation measured by MTT assay. Cells were incubated for 24 h with medium containing 0.1% FBS, and then treated with recombinant periostin with or without U0126. **** P
Figure Legend Snippet: Periostin promotes Ex3LL-cell proliferation and intracellular signaling in vitro (A) GFP fluorescence in Ex3LL-GFP cells co-cultured with periostin −/− or periostin +/+ fibroblasts ( n =5). Values on the Y-axis are normalized to the value on day 0. (B) Effect of periostin stimulation on Ex3LL proliferation, assayed by MTT ( n =5). Values on the Y-axis are normalized to the value on day 0. (C) Ex3LL cells were incubated for 24 h with medium containing 0.1% FBS and then treated with 1,000 ng/ml periostin for 5, 10, 30, or 60 min. The presence of ERK, AKT, FAK, and NF-κB and their phosphorylated forms [pERK (Thr202/Tyr204), pAKT (Thr308), pFAK (Tyr925), and p-NF-kB (Ser536)] in the Ex3LL cells was assessed by western blotting. Ratios of pERK to total ERK, based on band density analyzed with ImageJ software, are shown. (D) Ex3LL cell proliferation measured by MTT assay. Cells were incubated for 24 h with medium containing 0.1% FBS, and then treated with recombinant periostin with or without U0126. **** P

Techniques Used: In Vitro, Fluorescence, Cell Culture, MTT Assay, Incubation, Western Blot, Software, Recombinant

35) Product Images from "Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase C? and Extracellular Signal-regulated Kinase Signaling Pathways *"

Article Title: Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase C? and Extracellular Signal-regulated Kinase Signaling Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.162602

Knockdown and inhibition of PLD1 disrupt the activation of nuclear PKCα and ERK. A , HEK293 cells were transfected with siRNA for control or PLD1, treated with or without PDGF (10 ng/ml) for 30 min, fractionated into cytosol ( C ) and nucleus ( N
Figure Legend Snippet: Knockdown and inhibition of PLD1 disrupt the activation of nuclear PKCα and ERK. A , HEK293 cells were transfected with siRNA for control or PLD1, treated with or without PDGF (10 ng/ml) for 30 min, fractionated into cytosol ( C ) and nucleus ( N

Techniques Used: Inhibition, Activation Assay, Transfection

Nuclear localization of PLD1 is correlated with the activation of nuclear PKCα and ERK. A and B , HEK293 cells were transfected with the indicated GFP-PLD1 constructs, and the lysates were fractionated into cytosol ( C ) and nucleus ( N ) and analyzed
Figure Legend Snippet: Nuclear localization of PLD1 is correlated with the activation of nuclear PKCα and ERK. A and B , HEK293 cells were transfected with the indicated GFP-PLD1 constructs, and the lysates were fractionated into cytosol ( C ) and nucleus ( N ) and analyzed

Techniques Used: Activation Assay, Transfection, Construct

36) Product Images from "Isoalantolactone suppresses LPS-induced inflammation by inhibiting TRAF6 ubiquitination and alleviates acute lung injury"

Article Title: Isoalantolactone suppresses LPS-induced inflammation by inhibiting TRAF6 ubiquitination and alleviates acute lung injury

Journal: Acta Pharmacologica Sinica

doi: 10.1038/s41401-018-0061-3

IAL inhibits inflammation by deactivating NF-κB, MAPKs, and the Akt signaling pathway in BMDMs. a The protein expression levels of p-p65, p65, IκBα, p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-AKT, and AKT were evaluated by western blotting. BMDMs were pre-treated with IAL at 0, 2.5, 5, 10, and 20 μM for 30 min before stimulation with LPS (100 ng/mL) for 15 min (p-p65, p65, and IκBα) or 30 min (p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-AKT, and AKT). β-Actin was used as a loading control. b Statistical analysis of the above assay in histograms. The data represent the mean ± SEM of three independent experiments (* P
Figure Legend Snippet: IAL inhibits inflammation by deactivating NF-κB, MAPKs, and the Akt signaling pathway in BMDMs. a The protein expression levels of p-p65, p65, IκBα, p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-AKT, and AKT were evaluated by western blotting. BMDMs were pre-treated with IAL at 0, 2.5, 5, 10, and 20 μM for 30 min before stimulation with LPS (100 ng/mL) for 15 min (p-p65, p65, and IκBα) or 30 min (p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-AKT, and AKT). β-Actin was used as a loading control. b Statistical analysis of the above assay in histograms. The data represent the mean ± SEM of three independent experiments (* P

Techniques Used: Expressing, Western Blot

37) Product Images from "Shenmai injection suppresses multidrug resistance in MCF-7/ADR cells through the MAPK/NF-κB signalling pathway"

Article Title: Shenmai injection suppresses multidrug resistance in MCF-7/ADR cells through the MAPK/NF-κB signalling pathway

Journal: Pharmaceutical Biology

doi: 10.1080/13880209.2020.1742167

SMI reversed MDR of MCF-7/ADR cell via inhibition of MAPK/NF-κB pathway. (A,B) SMI inhibited the activation of MAPK pathway in MCF-7/ADR cells. (C,D) SMI inhibited the activation of NF-κB pathway in MCF-7/ADR cells. The protein level of p-ERK, p-JNK, P-IκBα, p-p65 was normalized to the level of tubulin, the loading control. Data are the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: SMI reversed MDR of MCF-7/ADR cell via inhibition of MAPK/NF-κB pathway. (A,B) SMI inhibited the activation of MAPK pathway in MCF-7/ADR cells. (C,D) SMI inhibited the activation of NF-κB pathway in MCF-7/ADR cells. The protein level of p-ERK, p-JNK, P-IκBα, p-p65 was normalized to the level of tubulin, the loading control. Data are the mean ± SEM of three independent experiments. * p

Techniques Used: Inhibition, Activation Assay

38) Product Images from "Myocilin Regulates Cell Proliferation and Survival *"

Article Title: Myocilin Regulates Cell Proliferation and Survival *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.547091

Inhibition of ERK activation abrogates myocilin effects on cell proliferation and survival. A , myocilin-expressing Tet-On HEK293 cells were pretreated with 10 μ m U0126 or DMSO for 2 h and further incubated in the medium containing or lacking 1
Figure Legend Snippet: Inhibition of ERK activation abrogates myocilin effects on cell proliferation and survival. A , myocilin-expressing Tet-On HEK293 cells were pretreated with 10 μ m U0126 or DMSO for 2 h and further incubated in the medium containing or lacking 1

Techniques Used: Inhibition, Activation Assay, Expressing, Incubation

Enhanced ERK activation in the trabecular meshwork of myocilin-expressing transgenic mice. A , Western blot analysis of the indicated proteins in the eye angle tissues. Lysates from the dissected angle tissues of 8-month-old wild-type or transgenic mice
Figure Legend Snippet: Enhanced ERK activation in the trabecular meshwork of myocilin-expressing transgenic mice. A , Western blot analysis of the indicated proteins in the eye angle tissues. Lysates from the dissected angle tissues of 8-month-old wild-type or transgenic mice

Techniques Used: Activation Assay, Expressing, Transgenic Assay, Mouse Assay, Western Blot

39) Product Images from "CD271 regulates the proliferation and motility of hypopharyngeal cancer cells"

Article Title: CD271 regulates the proliferation and motility of hypopharyngeal cancer cells

Journal: Scientific Reports

doi: 10.1038/srep30707

CD271-knockdown in HPCM2 cells reduces in vitro proliferation. ( A ) Proliferation of CD271-knockdown (siCD271#1 and siCD271#2) and control HPCM2 cells was assessed with MTT assays. ( n = 3) ( B ) HPCM2 cells were transfected with the siRNAs, incubated for 48 h, and then MYC expression profiles in CD271-knockdown and control HPCM2 cells were determined by real-time PCR. ( n = 3) ( C ) Cell cycle analysis of CD271-knockdown cells was performed by Ki67 and PI staining. The percentage of cells in G 0 is shown in the graph ( n = 3). ( D ) Western blot analysis of ERK, AKT, Iκbα, and p65 and their phosphorylated forms (p-ERK (Thr202/Tyr204), p-AKT (Ser473), p-Iκbα (Ser32), and pp65 (Ser536)) was performed with HPCM2 cells incubated for 24 h in serum-free medium and then treated with 100 ng/ml NGF for 10 min. p-ERK/ERK, the ratio of p-ERK to total ERK based on band density analysis with ImageJ software. ( E ) HPCM2 proliferation assays after treatment with U0126. The relative intensity values represent the ratio of the MTT absorbance after the cells were incubated for 72 h with U0126 to that measured at 0 h of treatment. ( n = 4) ( F ) ERK phosphorylation was analyzed by Western blotting after 72 hours of U0126 treatment. PI, propidium iodide.
Figure Legend Snippet: CD271-knockdown in HPCM2 cells reduces in vitro proliferation. ( A ) Proliferation of CD271-knockdown (siCD271#1 and siCD271#2) and control HPCM2 cells was assessed with MTT assays. ( n = 3) ( B ) HPCM2 cells were transfected with the siRNAs, incubated for 48 h, and then MYC expression profiles in CD271-knockdown and control HPCM2 cells were determined by real-time PCR. ( n = 3) ( C ) Cell cycle analysis of CD271-knockdown cells was performed by Ki67 and PI staining. The percentage of cells in G 0 is shown in the graph ( n = 3). ( D ) Western blot analysis of ERK, AKT, Iκbα, and p65 and their phosphorylated forms (p-ERK (Thr202/Tyr204), p-AKT (Ser473), p-Iκbα (Ser32), and pp65 (Ser536)) was performed with HPCM2 cells incubated for 24 h in serum-free medium and then treated with 100 ng/ml NGF for 10 min. p-ERK/ERK, the ratio of p-ERK to total ERK based on band density analysis with ImageJ software. ( E ) HPCM2 proliferation assays after treatment with U0126. The relative intensity values represent the ratio of the MTT absorbance after the cells were incubated for 72 h with U0126 to that measured at 0 h of treatment. ( n = 4) ( F ) ERK phosphorylation was analyzed by Western blotting after 72 hours of U0126 treatment. PI, propidium iodide.

Techniques Used: In Vitro, MTT Assay, Transfection, Incubation, Expressing, Real-time Polymerase Chain Reaction, Cell Cycle Assay, Staining, Western Blot, Software

40) Product Images from "PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition"

Article Title: PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks760

Model of regulating IRES-mediated LamB1 translation during hepatocellular EMT and carcinoma progression. PDGF signaling induced by TGF-β activates the MAPK/ERK pathway that predominantly regulates the cytoplasmic accumulation of La. PI3K/AKT might be additionally involved in the translocation of La to the cytoplasm (dashed lines). La binds as an ITAF to the minimal IRES motif and enhances IRES-mediated translation of LamB1 in EMT-transformed cells. Winded arrows indicate autocrine regulation of TGF-β and PDGF (top).
Figure Legend Snippet: Model of regulating IRES-mediated LamB1 translation during hepatocellular EMT and carcinoma progression. PDGF signaling induced by TGF-β activates the MAPK/ERK pathway that predominantly regulates the cytoplasmic accumulation of La. PI3K/AKT might be additionally involved in the translocation of La to the cytoplasm (dashed lines). La binds as an ITAF to the minimal IRES motif and enhances IRES-mediated translation of LamB1 in EMT-transformed cells. Winded arrows indicate autocrine regulation of TGF-β and PDGF (top).

Techniques Used: Translocation Assay, Transformation Assay

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Article Snippet: .. About 25 μg-50 μg protein extracts were electrophoresed on 10-15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto PVDF membranes (0.45 μm, Millipore, BioRad), which were then blocked with 5% BSA in TBST for 1 h, and incubated overnight at 4°C with following primary antibodies: anti-Bax, anti-Bcl-2, anti-p-Akt (pSer473), anti-Akt, anti-p-ERK, anti-ERK, anti-PARP and anti-caspase 3 (1:1000, Cell Signaling Technology, USA). .. After washing 3 times with TBST, the membranes were incubated for 2 h at room temperature with secondary HRP-conjugated sheep anti-rabbit antibody or HRP-conjugated sheep anti-mouse antibody diluted in TBST with 5% BSA (1:2000, Santa Cruz Biotech, USA).

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Article Title: Plumbagin inhibits tumour angiogenesis and tumour growth through the Ras signalling pathway following activation of the VEGF receptor-2
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SDS Page:

Article Title: Magnesium isoglycyrrhizinate protects hepatic L02 cells from ischemia/reperfusion induced injury
Article Snippet: .. About 25 μg-50 μg protein extracts were electrophoresed on 10-15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto PVDF membranes (0.45 μm, Millipore, BioRad), which were then blocked with 5% BSA in TBST for 1 h, and incubated overnight at 4°C with following primary antibodies: anti-Bax, anti-Bcl-2, anti-p-Akt (pSer473), anti-Akt, anti-p-ERK, anti-ERK, anti-PARP and anti-caspase 3 (1:1000, Cell Signaling Technology, USA). .. After washing 3 times with TBST, the membranes were incubated for 2 h at room temperature with secondary HRP-conjugated sheep anti-rabbit antibody or HRP-conjugated sheep anti-mouse antibody diluted in TBST with 5% BSA (1:2000, Santa Cruz Biotech, USA).

Blocking Assay:

Article Title: Shenmai injection suppresses multidrug resistance in MCF-7/ADR cells through the MAPK/NF-κB signalling pathway
Article Snippet: .. Then, 5% skim milk was used to block PVDF membranes at room temperature for 2 h. Afterwards, the membranes were probed with primary antibodies anti-P-gp, anti-JNK, anti-ERK, anti-p38, a nti-p65 (diluted 1:1000, CST, Beverly, MA, USA), anti-IκBα or anti-tubulin (diluted 1:1000, Santa Cruz Biotechnology, DBA Italy) at 4 °C overnight. .. After being washed with Tris-buffered saline/Tween-20 (TBS/T), the membranes were incubated with secondary antibody horseradish peroxidase conjugated goat anti-mouse IgG or goat anti-rabbit IgG (diluted 1:2000, Bioker Biological Technology, China) for 2 h at room temperature.

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    Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 <t>ERK,</t> phospho-p42/44 ERK, <t>total-AKT,</t> phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.
    Anti Total Extracellular Signal Related Kinase Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Representative immunoblots and densitometric analyses of total ERK 1/2, Thr 202/ Tyr-204 P-ERK 1/2, total CREB, and Ser-133 P-CREB in the border zone of hearts exposed to ischemia-reperfusion. The myocardium of rats that were not exposed to ischemia-reperfusion and received vehicle served as the control. β-actin was used as a loading control. *  P
    Anti Thr 202 Tyr 204 P Erk 1 2, supplied by Cell Signaling Technology Inc, 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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    Cell Signaling Technology Inc erk antibody
    Signaling mechanisms involved in Lin28a stimulation. ( A ) Western blot analysis of the expression of AKT, p-AKT, <t>ERK,</t> p-ERK, mTOR, p-mTOR, S6, p-S6, and CXCR4. ( B ) A schematic of how Lin28a may affect the self-renewal of mGSCs. Lin28a activated Gfra1 and then activated the AKT signal pathway. Then, AKT activated the mTOR-S6 pathway to promote the expression of proliferation-related genes to maintain self-renewal or activate the expression of <t>ETV5</t> to directly maintain self-renewal. By activating CXCR4, Lin28a activated the ERK pathway to promote the expression of proliferation-related genes to maintain self-renewal. **p
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    Image Search Results


    Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Journal: BioMed Research International

    Article Title: Degalactotigonin, a Steroidal Glycoside from Solanum nigrum, Induces Apoptosis and Cell Cycle Arrest via Inhibiting the EGFR Signaling Pathways in Pancreatic Cancer Cells

    doi: 10.1155/2018/3120972

    Figure Lengend Snippet: Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Article Snippet: Anti-phospho-EGFR, anti-Akt, anti-phospho Akt (Ser473), anti-ERK, and anti-phospho-ERK antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Western Blot, Expressing, Software

    Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 ERK, phospho-p42/44 ERK, total-AKT, phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.

    Journal: Annals of Dermatology

    Article Title: Role of Arachidonic Acid in Promoting Hair Growth

    doi: 10.5021/ad.2016.28.1.55

    Figure Lengend Snippet: Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 ERK, phospho-p42/44 ERK, total-AKT, phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.

    Article Snippet: The blotted membranes were incubated at 4℃ with the appropriate antibodies: anti-total extracellular signal-related kinase (ERK), anti-phosphorylated ERK, anti-total protein kinase B (Akt), anti-phosphorylated AKT, anti-B-cell lymphoma 2 (Bcl-2), anti-total cyclic AMP response element-binding protein (CREB), anti-phosphorylated CREB (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot, Binding Assay, CTL Assay

    Representative immunoblots and densitometric analyses of total ERK 1/2, Thr 202/ Tyr-204 P-ERK 1/2, total CREB, and Ser-133 P-CREB in the border zone of hearts exposed to ischemia-reperfusion. The myocardium of rats that were not exposed to ischemia-reperfusion and received vehicle served as the control. β-actin was used as a loading control. *  P

    Journal: Cardiovascular Drugs and Therapy

    Article Title: Dipyridamole with Low-Dose Aspirin Augments the Infarct Size-Limiting Effects of Simvastatin

    doi: 10.1007/s10557-010-6252-x

    Figure Lengend Snippet: Representative immunoblots and densitometric analyses of total ERK 1/2, Thr 202/ Tyr-204 P-ERK 1/2, total CREB, and Ser-133 P-CREB in the border zone of hearts exposed to ischemia-reperfusion. The myocardium of rats that were not exposed to ischemia-reperfusion and received vehicle served as the control. β-actin was used as a loading control. * P

    Article Snippet: Anti-eNOS, anti-Ser-1177 P-eNOS, anti-ERK 1/2, anti-Thr-202/Tyr-204 P-ERK 1/2, anti-CREB, and anti-Ser-133 P-CREB antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

    Techniques: Western Blot

    Signaling mechanisms involved in Lin28a stimulation. ( A ) Western blot analysis of the expression of AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, S6, p-S6, and CXCR4. ( B ) A schematic of how Lin28a may affect the self-renewal of mGSCs. Lin28a activated Gfra1 and then activated the AKT signal pathway. Then, AKT activated the mTOR-S6 pathway to promote the expression of proliferation-related genes to maintain self-renewal or activate the expression of ETV5 to directly maintain self-renewal. By activating CXCR4, Lin28a activated the ERK pathway to promote the expression of proliferation-related genes to maintain self-renewal. **p

    Journal: Scientific Reports

    Article Title: Lin28a promotes self-renewal and proliferation of dairy goat spermatogonial stem cells (SSCs) through regulation of mTOR and PI3K/AKT

    doi: 10.1038/srep38805

    Figure Lengend Snippet: Signaling mechanisms involved in Lin28a stimulation. ( A ) Western blot analysis of the expression of AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, S6, p-S6, and CXCR4. ( B ) A schematic of how Lin28a may affect the self-renewal of mGSCs. Lin28a activated Gfra1 and then activated the AKT signal pathway. Then, AKT activated the mTOR-S6 pathway to promote the expression of proliferation-related genes to maintain self-renewal or activate the expression of ETV5 to directly maintain self-renewal. By activating CXCR4, Lin28a activated the ERK pathway to promote the expression of proliferation-related genes to maintain self-renewal. **p

    Article Snippet: The membranes were incubated with primary antibodies, including the LIN28A antibody (1:500, Santa Cruz), SOX2 antibody (1:200, Bioss), PCNA antibody (1:1000, Biolegend), OCT4 antibody (1:200, Bioss), ETV5 antibody (1:1000, abcam), GFRA1 antibody (1:400, Santa Cruz), ERK antibody (1:1000, Cell Signaling Technology), p-ERK antibody (1:500, Cell Signaling Technology), AKT antibody (1:1000, BOSTER, Wuhan, China), p-AKT antibody (1:500, Sangon), S6 antibody (1:1000, Cell Signaling Technology), mTOR antibody (1:1000, Cell Signaling Technology), p-mTOR antibody (Ser2448, 1:1000, Cell Signaling Technology), pS6 (Ser235/236, 1:1000, Cell Signaling Technology), CXCR4 antibody (1:400, Bioss), and anti-β-actin antibody (1:1,000, Cell Signaling Technology).

    Techniques: Western Blot, Expressing