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  • 99
    Name:
    PathScan Total Smad2 3 Sandwich ELISA Kit
    Description:
    The PathScan Total Smad2 3 Sandwich ELISA Kit is a solid phase sandwich enzyme linked immunosorbent assay ELISA that recognizes endogenous levels of Smad2 and Smad3 proteins A Smad2 3 Mouse Antibody has been coated on the microwells After incubation with cell lysates Smad2 3 proteins phospho and nonphospho are captured by the coated antibody Following extensive washing a Smad2 3 Rabbit Detection Antibody is added to detect captured Smad2 3 proteins Anti rabbit IgG HRP linked Antibody is then used to recognize the bound detection antibody HRP substrate TMB is added to develop color The magnitude of the absorbance for this developed color is proportional to the quantity of Smad2 and Smad3 proteins
    Catalog Number:
    12000
    Price:
    None
    Category:
    ELISA Kits
    Reactivity:
    Human Mouse Mink
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc erk
    P38 MAPK and <t>ERK</t> are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and <t>JNK</t> were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P
    The PathScan Total Smad2 3 Sandwich ELISA Kit is a solid phase sandwich enzyme linked immunosorbent assay ELISA that recognizes endogenous levels of Smad2 and Smad3 proteins A Smad2 3 Mouse Antibody has been coated on the microwells After incubation with cell lysates Smad2 3 proteins phospho and nonphospho are captured by the coated antibody Following extensive washing a Smad2 3 Rabbit Detection Antibody is added to detect captured Smad2 3 proteins Anti rabbit IgG HRP linked Antibody is then used to recognize the bound detection antibody HRP substrate TMB is added to develop color The magnitude of the absorbance for this developed color is proportional to the quantity of Smad2 and Smad3 proteins
    https://www.bioz.com/result/erk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1210 article reviews
    Price from $9.99 to $1999.99
    erk - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs"

    Article Title: Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201712113

    P38 MAPK and ERK are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and JNK were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P
    Figure Legend Snippet: P38 MAPK and ERK are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and JNK were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P

    Techniques Used: Activation Assay, Cell Culture

    2) Product Images from "RAF dimer inhibition enhances the antitumor activity of MEK inhibitors in K‐RAS mutant tumors"

    Article Title: RAF dimer inhibition enhances the antitumor activity of MEK inhibitors in K‐RAS mutant tumors

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12698

    Effect of different allosteric MEKi combined with BGB‐283 and their impact on the MEK/ERK pathway in Calu‐6 cells. The antiproliferative effect of combining BGB‐283 and (A) PD‐0325901, (B) pimasertib, (C) trametinib, or (D) RO5126766 was evaluated by EOHSA analysis. The considered dose combinations with synergy found by Pacifico’s approach at significance level 0.05 are highlighted. (E) Immunoblot for pMEK, MEK, pERK, ERK, and GAPDH in Calu‐6 cells after 1‐ and 24‐h treatment with DMSO (‐), selumetinib, PD‐0325901, pimasertib, trametinib, and RO5126766 at the indicated concentrations. (F) Dose–response curve of MEK phosphorylation was determined by HTRF assay after 1‐h treatment of serial dilutions of different MEKi in Calu‐6 cells.
    Figure Legend Snippet: Effect of different allosteric MEKi combined with BGB‐283 and their impact on the MEK/ERK pathway in Calu‐6 cells. The antiproliferative effect of combining BGB‐283 and (A) PD‐0325901, (B) pimasertib, (C) trametinib, or (D) RO5126766 was evaluated by EOHSA analysis. The considered dose combinations with synergy found by Pacifico’s approach at significance level 0.05 are highlighted. (E) Immunoblot for pMEK, MEK, pERK, ERK, and GAPDH in Calu‐6 cells after 1‐ and 24‐h treatment with DMSO (‐), selumetinib, PD‐0325901, pimasertib, trametinib, and RO5126766 at the indicated concentrations. (F) Dose–response curve of MEK phosphorylation was determined by HTRF assay after 1‐h treatment of serial dilutions of different MEKi in Calu‐6 cells.

    Techniques Used: HTRF Assay

    Combination of BGB‐283 and selumetinib effectively inhibited selumetinib‐induced pMEK accumulation and led to sustained pERK reduction. (A) Dose–response curve of MEK phosphorylation in Calu‐6 cells treated with 1 µ m selumetinib combined with increasing concentrations of BGB‐283 or vemurafenib for 1, 6, and 24 h. (B) Immunoblotting of B‐RAF, C‐RAF, pMEK, MEK, pERK, and ERK in Calu‐6 cells incubated with 1 µ m selumetinib alone or combined with 1 µ m BGB‐283 or 1 µ m vemurafenib after 1‐ to 48‐h treatment. (C) Biochemical activity of compound C against WT B‐RAF, B‐RAF V600E , C‐RAF, and EGFR. (D) Comparison of synergistic effect of MEKi with BGB‐283 or compound C from P ‐value, the percentage of dose combination with synergy, and maximum EC 50 shift in Calu‐6 cells.
    Figure Legend Snippet: Combination of BGB‐283 and selumetinib effectively inhibited selumetinib‐induced pMEK accumulation and led to sustained pERK reduction. (A) Dose–response curve of MEK phosphorylation in Calu‐6 cells treated with 1 µ m selumetinib combined with increasing concentrations of BGB‐283 or vemurafenib for 1, 6, and 24 h. (B) Immunoblotting of B‐RAF, C‐RAF, pMEK, MEK, pERK, and ERK in Calu‐6 cells incubated with 1 µ m selumetinib alone or combined with 1 µ m BGB‐283 or 1 µ m vemurafenib after 1‐ to 48‐h treatment. (C) Biochemical activity of compound C against WT B‐RAF, B‐RAF V600E , C‐RAF, and EGFR. (D) Comparison of synergistic effect of MEKi with BGB‐283 or compound C from P ‐value, the percentage of dose combination with synergy, and maximum EC 50 shift in Calu‐6 cells.

    Techniques Used: Incubation, Activity Assay

    3) Product Images from "SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression"

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2015.23

    Knockdown of SPINK1 reduces PI3K/AKT and MEK/ERK signaling. ( a ) AKT and ERK phosphorylation was determined by western blotting using shSPINK1-1, shSPINK1-2, shSPINK1-3 and shSCRM WiDr cells. ( b ) Western blotting for AKT, MEK and ERK phosphorylation for WiDr cells treated with 5 and 10 μ M of AKT inhibitor (LY294002) and MEK inhibitor (PD98059) and DMSO control. ( c ) Cell proliferation assay using WiDr cells treated with 5 , 10 and 25 μ M of AKT inhibitor (LY294002) with DMSO as control. ( d ) Same as c , except MEK inhibitor (PD98059) was used. ( e ) Foci formation assay using WiDr cells treated with AKT and MEK inhibitor. ( f ) Boyden chamber matrigel invasion assay using WiDr cells treated with 10 and 25 μ M of AKT inhibitor (LY294002) and DMSO. ( g ) Same as f , except cells were treated with MEK inhibitor (PD98059). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P
    Figure Legend Snippet: Knockdown of SPINK1 reduces PI3K/AKT and MEK/ERK signaling. ( a ) AKT and ERK phosphorylation was determined by western blotting using shSPINK1-1, shSPINK1-2, shSPINK1-3 and shSCRM WiDr cells. ( b ) Western blotting for AKT, MEK and ERK phosphorylation for WiDr cells treated with 5 and 10 μ M of AKT inhibitor (LY294002) and MEK inhibitor (PD98059) and DMSO control. ( c ) Cell proliferation assay using WiDr cells treated with 5 , 10 and 25 μ M of AKT inhibitor (LY294002) with DMSO as control. ( d ) Same as c , except MEK inhibitor (PD98059) was used. ( e ) Foci formation assay using WiDr cells treated with AKT and MEK inhibitor. ( f ) Boyden chamber matrigel invasion assay using WiDr cells treated with 10 and 25 μ M of AKT inhibitor (LY294002) and DMSO. ( g ) Same as f , except cells were treated with MEK inhibitor (PD98059). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

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

    4) Product Images from "Increased BMPR1A Expression Enhances the Adipogenic Differentiation of Mesenchymal Stem Cells in Patients with Ankylosing Spondylitis"

    Article Title: Increased BMPR1A Expression Enhances the Adipogenic Differentiation of Mesenchymal Stem Cells in Patients with Ankylosing Spondylitis

    Journal: Stem Cells International

    doi: 10.1155/2019/4143167

    ASMSCs had higher pSmad1/5/8 expression during adipogenesis. The levels of proteins involved in adipogenic-related signaling pathways were assessed. (a) Higher levels of the pSmad1/5/8 proteins were detected in ASMSCs ( n = 25) than in HDMSCs ( n = 30) on days 7, 10, 14, and 21. (b) No differences in the active β -catenin/ β -catenin, p-CREB/CREB, p-AKT/AKT, p-ERK/ERK, p-p38/p38, or p-JNK/JNK ratios were observed between HDMSCs and ASMSCs. Values are presented as the means ± SDs. ∗ P
    Figure Legend Snippet: ASMSCs had higher pSmad1/5/8 expression during adipogenesis. The levels of proteins involved in adipogenic-related signaling pathways were assessed. (a) Higher levels of the pSmad1/5/8 proteins were detected in ASMSCs ( n = 25) than in HDMSCs ( n = 30) on days 7, 10, 14, and 21. (b) No differences in the active β -catenin/ β -catenin, p-CREB/CREB, p-AKT/AKT, p-ERK/ERK, p-p38/p38, or p-JNK/JNK ratios were observed between HDMSCs and ASMSCs. Values are presented as the means ± SDs. ∗ P

    Techniques Used: Expressing

    5) Product Images from "IL-22 Confers EGFR-TKI Resistance in NSCLC via the AKT and ERK Signaling Pathways"

    Article Title: IL-22 Confers EGFR-TKI Resistance in NSCLC via the AKT and ERK Signaling Pathways

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.01167

    IL-22 promoted activation of the phosphorylated EGFR/AKT/ERK in NSCLC cells. (A) PC-9 and (C) HCC827 cells were incubated in the presence or absence of gefitinib (IC50) for 3 h prior to exposure to 50 ng/ml IL-22 (+/−), 10 4 ng/mL IL-22 neutralizing antibody (+/−) and mouse IgG 1 Isotype control (+/−) for additional 24 h, and the protein samples were analyzed by western blot with indicated antibodies. (B) PC-9 and (D) HCC827 cells densitometric analysis of western blots for the phosphorylated protein. * P
    Figure Legend Snippet: IL-22 promoted activation of the phosphorylated EGFR/AKT/ERK in NSCLC cells. (A) PC-9 and (C) HCC827 cells were incubated in the presence or absence of gefitinib (IC50) for 3 h prior to exposure to 50 ng/ml IL-22 (+/−), 10 4 ng/mL IL-22 neutralizing antibody (+/−) and mouse IgG 1 Isotype control (+/−) for additional 24 h, and the protein samples were analyzed by western blot with indicated antibodies. (B) PC-9 and (D) HCC827 cells densitometric analysis of western blots for the phosphorylated protein. * P

    Techniques Used: Activation Assay, Incubation, Western Blot

    6) Product Images from "Polyphyllin VI induces apoptosis and autophagy in human osteosarcoma cells by modulation of ROS/JNK activation"

    Article Title: Polyphyllin VI induces apoptosis and autophagy in human osteosarcoma cells by modulation of ROS/JNK activation

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S194961

    Intracellular H2O2 and JNK activation involved in Polyphyllin VI-induced autophagy and apoptosis. ( A ) U2OS cells were incubated with the indicated concentrations of Polyphyllin VI for 24 h. The H2O2 level was measured by flow cytometry analysis. ( B ) Cells were treated with 7.5 μM Polyphylliin VI alone or in combination with the ROS scavenger NAC (10 mM), and then cells were subjected to flow cytometry analysis of intracellular H2O2. ( C ) Western blot analysis of p-JNK, JNK, p-ERK, ERK, p-p38 and p38 in cells treated with Polyphyllin VI at the indicated doses for 24 h. *P
    Figure Legend Snippet: Intracellular H2O2 and JNK activation involved in Polyphyllin VI-induced autophagy and apoptosis. ( A ) U2OS cells were incubated with the indicated concentrations of Polyphyllin VI for 24 h. The H2O2 level was measured by flow cytometry analysis. ( B ) Cells were treated with 7.5 μM Polyphylliin VI alone or in combination with the ROS scavenger NAC (10 mM), and then cells were subjected to flow cytometry analysis of intracellular H2O2. ( C ) Western blot analysis of p-JNK, JNK, p-ERK, ERK, p-p38 and p38 in cells treated with Polyphyllin VI at the indicated doses for 24 h. *P

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Western Blot

    7) Product Images from "Genetic and pharmacological activation of Hedgehog signaling inhibits osteoclastogenesis and attenuates titanium particle-induced osteolysis partly through suppressing the JNK/c-Fos-NFATc1 cascade"

    Article Title: Genetic and pharmacological activation of Hedgehog signaling inhibits osteoclastogenesis and attenuates titanium particle-induced osteolysis partly through suppressing the JNK/c-Fos-NFATc1 cascade

    Journal: Theranostics

    doi: 10.7150/thno.44793

    Activation of Hh signaling inhibited RANKL-induced activation of JNK pathway. (A) Western blot analyses of protein levels of phosphorylated ERK (p-ERK), total ERK, phosphorylated JNK (p-JNK), total JNK, phosphorylated p38 (p-p38), and total p38 in BMMs treated with 50 ng/ml RANKL in the presence of vehicle (Veh) or 2 µM PM (PM) for indicate times. Representative images from three independent biological replicates were shown. (B-D) Quantitative analyses of relative ratios of p-ERK/ERK (B), p-JNK/JNK(C) and p-p38/p38 (D). n=3 per group. All bar graphs were presented as mean ± SD. * P
    Figure Legend Snippet: Activation of Hh signaling inhibited RANKL-induced activation of JNK pathway. (A) Western blot analyses of protein levels of phosphorylated ERK (p-ERK), total ERK, phosphorylated JNK (p-JNK), total JNK, phosphorylated p38 (p-p38), and total p38 in BMMs treated with 50 ng/ml RANKL in the presence of vehicle (Veh) or 2 µM PM (PM) for indicate times. Representative images from three independent biological replicates were shown. (B-D) Quantitative analyses of relative ratios of p-ERK/ERK (B), p-JNK/JNK(C) and p-p38/p38 (D). n=3 per group. All bar graphs were presented as mean ± SD. * P

    Techniques Used: Activation Assay, Western Blot

    8) Product Images from "Cimifugin ameliorates imiquimod-induced psoriasis by inhibiting oxidative stress and inflammation via NF-κB/MAPK pathway"

    Article Title: Cimifugin ameliorates imiquimod-induced psoriasis by inhibiting oxidative stress and inflammation via NF-κB/MAPK pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20200471

    Cimifugin suppressed TNF-α-induced activation of NF-кB and MAPK signaling pathways in HaCaT cells ( A – C ) The expression levels of p-IкB, IкB, p-p65, and p65 proteins for NF-кB signaling pathway were detected by western blot. ( D ) The activation of p65 was evaluated using immnofluorescence staining. ( E – H ) The expression levels of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 proteins for MAPK signaling pathway were tested by western blot. CIM-L, low dose of cimifugin; CIM-M, medium dose of cimifugin; CIM-H, high dose of cimifugin. ## P
    Figure Legend Snippet: Cimifugin suppressed TNF-α-induced activation of NF-кB and MAPK signaling pathways in HaCaT cells ( A – C ) The expression levels of p-IкB, IкB, p-p65, and p65 proteins for NF-кB signaling pathway were detected by western blot. ( D ) The activation of p65 was evaluated using immnofluorescence staining. ( E – H ) The expression levels of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 proteins for MAPK signaling pathway were tested by western blot. CIM-L, low dose of cimifugin; CIM-M, medium dose of cimifugin; CIM-H, high dose of cimifugin. ## P

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

    Cimifugin suppressed the activation of NF-кB and MAPK signaling pathways in IMQ-induced mice ( A – C ) The expression levels of p-IкB, IкB, p-p65, and p65 proteins for NF-кB signaling pathway were detected by western blot. ( D – G ) The expression levels of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 proteins for MAPK signaling pathway were tested by western blot. CIM-L, low dose of cimifugin; CIM-H, high dose of cimifugin. ## P
    Figure Legend Snippet: Cimifugin suppressed the activation of NF-кB and MAPK signaling pathways in IMQ-induced mice ( A – C ) The expression levels of p-IкB, IкB, p-p65, and p65 proteins for NF-кB signaling pathway were detected by western blot. ( D – G ) The expression levels of p-JNK, JNK, p-ERK, ERK, p-p38, and p38 proteins for MAPK signaling pathway were tested by western blot. CIM-L, low dose of cimifugin; CIM-H, high dose of cimifugin. ## P

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

    9) Product Images from "Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy"

    Article Title: Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2016.143

    p38 MAPK induced an autophagy-dependent pathway in ASA-regulated CF proliferation. (A) CFs exposed to hypoxia were treated with doses of ASA at 0.5, 2.5, and 5 mmol/L for 12 h, and then the changes of phosphorylated p38, ERK, JNK were detected by Western blot. (B) Cardiac fibroblasts were pre-treated with the p38 inhibitor SB203580 (10 μmol/L) for 1 h and were then treated with 2.5 mmol/L ASA for 12 h. Western blot results showed the changes of LC3-II, phosphorylated Akt and cleaved PARP 1. Mean±SEM. n =3, ** P
    Figure Legend Snippet: p38 MAPK induced an autophagy-dependent pathway in ASA-regulated CF proliferation. (A) CFs exposed to hypoxia were treated with doses of ASA at 0.5, 2.5, and 5 mmol/L for 12 h, and then the changes of phosphorylated p38, ERK, JNK were detected by Western blot. (B) Cardiac fibroblasts were pre-treated with the p38 inhibitor SB203580 (10 μmol/L) for 1 h and were then treated with 2.5 mmol/L ASA for 12 h. Western blot results showed the changes of LC3-II, phosphorylated Akt and cleaved PARP 1. Mean±SEM. n =3, ** P

    Techniques Used: Western Blot

    10) Product Images from "Baicalin induces cellular senescence in human colon cancer cells via upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling"

    Article Title: Baicalin induces cellular senescence in human colon cancer cells via upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0223-0

    Ras/Raf/MEK/ERK signaling pathway was involved in DEPP-mediated tumor cellular senescence in colon cancer cells. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with siRNA targeting Raf1 and ERK, then immunoblotting analysis a and SA-β-Gal staining c were carried out. Shown are representative of 4 experiments. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with Sorafenib or U0126 for 24 h, then immunoblotting analysis b , SA-β-Gal staining d , EdU staining e and colony formation assay f were carried out. Shown are representative of 4 experiments. The quantification of SA-β-Gal and crystal violet staining represents the means ± SD. The integrated optical density of EdU staining represents the means ± SD. (*** P ≤ 0.001 versus the control group). g Immunoblotting of Flag-DEPP immunoprecipitates from HCT116 cells with or without the treatment with baicalin or siDEPP. The binding of DEPP to Ras in cells is presented relative to that in the untreated cells. Shown are representative of three experiments
    Figure Legend Snippet: Ras/Raf/MEK/ERK signaling pathway was involved in DEPP-mediated tumor cellular senescence in colon cancer cells. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with siRNA targeting Raf1 and ERK, then immunoblotting analysis a and SA-β-Gal staining c were carried out. Shown are representative of 4 experiments. HCT116 cells transfected with pcDNA3.1 either blank or loaded the cDNA of DEPP were treated with Sorafenib or U0126 for 24 h, then immunoblotting analysis b , SA-β-Gal staining d , EdU staining e and colony formation assay f were carried out. Shown are representative of 4 experiments. The quantification of SA-β-Gal and crystal violet staining represents the means ± SD. The integrated optical density of EdU staining represents the means ± SD. (*** P ≤ 0.001 versus the control group). g Immunoblotting of Flag-DEPP immunoprecipitates from HCT116 cells with or without the treatment with baicalin or siDEPP. The binding of DEPP to Ras in cells is presented relative to that in the untreated cells. Shown are representative of three experiments

    Techniques Used: Transfection, Staining, Colony Assay, Binding Assay

    Baicalin upregulated DEPP expression and induced senescence in colon cancer cells in vivo. a , b Effects of baicalin on tumor volume and body weight. The data are representative of results obtained from 5 mice. c Tumor sections were subjected to hematoxylin and eosin staining, SA-β-Gal staining and IHC for Ki67, p16 INK4A and cleaved-caspase 3. Original magnification was 400×. Every single shown picture is representative of five sections from every group. Different sections represent different tumors. The representative images and the calculation of the percentage of positive cells are shown in d – g ; the data are the means ± SD (*** P ≤ 0.001). h Tumors were collected for western blot analysis of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin. Every single lane represents one tumor from an individual mouse. i Tumor sections were subjected to cleaved-caspase 3. Original magnification was 400×
    Figure Legend Snippet: Baicalin upregulated DEPP expression and induced senescence in colon cancer cells in vivo. a , b Effects of baicalin on tumor volume and body weight. The data are representative of results obtained from 5 mice. c Tumor sections were subjected to hematoxylin and eosin staining, SA-β-Gal staining and IHC for Ki67, p16 INK4A and cleaved-caspase 3. Original magnification was 400×. Every single shown picture is representative of five sections from every group. Different sections represent different tumors. The representative images and the calculation of the percentage of positive cells are shown in d – g ; the data are the means ± SD (*** P ≤ 0.001). h Tumors were collected for western blot analysis of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin. Every single lane represents one tumor from an individual mouse. i Tumor sections were subjected to cleaved-caspase 3. Original magnification was 400×

    Techniques Used: Expressing, In Vivo, Mouse Assay, Staining, Immunohistochemistry, Western Blot

    DEPP was upregulated by baicalin in cancer cells in vitro. a HCT116 cells treated with vehicle or baicalin were subjected to whole-genome microarray analysis. The histogram depicts a compilation of hypoxia-response genes. b The expression of DEPP mRNA in the vehicle-treated or baicalin-treated cells was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. c HCT116 cells were exposed to hypoxic condition for indicated times, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin were analyzed by western blotting. d The expression of DEPP mRNA in HCT116 cells exposed to hypoxic condition was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. e HCT116 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK and β-actin were analyzed by Western blotting. f HCT116 cells were treated with vehicle or varying concentrations of baicalin (10, 20 and 40 μM) for 24 h, and the protein levels of p16 INK4A , pRb, Rb, p21, p27, cleaved-caspase 3 and caspase 3 were analyzed by western blotting. g , h A549 and Panc-1 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin were analyzed by Western blotting. (*** P ≤ 0.001 versus the control group)
    Figure Legend Snippet: DEPP was upregulated by baicalin in cancer cells in vitro. a HCT116 cells treated with vehicle or baicalin were subjected to whole-genome microarray analysis. The histogram depicts a compilation of hypoxia-response genes. b The expression of DEPP mRNA in the vehicle-treated or baicalin-treated cells was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. c HCT116 cells were exposed to hypoxic condition for indicated times, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin were analyzed by western blotting. d The expression of DEPP mRNA in HCT116 cells exposed to hypoxic condition was verified by qRT–PCR. The data shown are means ± SD from three independent experiments. e HCT116 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK and β-actin were analyzed by Western blotting. f HCT116 cells were treated with vehicle or varying concentrations of baicalin (10, 20 and 40 μM) for 24 h, and the protein levels of p16 INK4A , pRb, Rb, p21, p27, cleaved-caspase 3 and caspase 3 were analyzed by western blotting. g , h A549 and Panc-1 cells were treated with vehicle or baicalin at indicated concentrations, and the protein levels of DEPP, p-Raf1, Raf1, p-ERK, ERK, p16 INK4A , pRb, Rb and β-actin were analyzed by Western blotting. (*** P ≤ 0.001 versus the control group)

    Techniques Used: In Vitro, Microarray, Expressing, Quantitative RT-PCR, Western Blot

    11) Product Images from "Inhibition of p38? MAPK rescues cardiomyopathy induced by overexpressed ?2-adrenergic receptor, but not ?1-adrenergic receptor"

    Article Title: Inhibition of p38? MAPK rescues cardiomyopathy induced by overexpressed ?2-adrenergic receptor, but not ?1-adrenergic receptor

    Journal:

    doi: 10.1172/JCI29576

    ERK and JNK.
    Figure Legend Snippet: ERK and JNK.

    Techniques Used:

    12) Product Images from "Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases"

    Article Title: Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases

    Journal: Scientific Reports

    doi: 10.1038/srep13824

    CA attenuates ADP-activated platelet p38MAPK, ERK and JNK phosphorylation. Washed platelets were incubated with CA or vehicle for 20 min and then stimulated with ADP for 20 min. Platelet proteins were then extracted and specific antibodies were used to measure the levels of total and phosphorylated p38 ( A ), ERK ( B ), JNK ( C ), and Akt ( D ). CA attenuated the phosphorylation of p38, ERK and JNK but not of Akt (Ser473) in the activated platelets. Images are representative of three separate experiments. The relative protein expression levels were quantified by Quantity One software. Data represent the mean ± SEM of at least three independent experiments performed in triplicate. * p
    Figure Legend Snippet: CA attenuates ADP-activated platelet p38MAPK, ERK and JNK phosphorylation. Washed platelets were incubated with CA or vehicle for 20 min and then stimulated with ADP for 20 min. Platelet proteins were then extracted and specific antibodies were used to measure the levels of total and phosphorylated p38 ( A ), ERK ( B ), JNK ( C ), and Akt ( D ). CA attenuated the phosphorylation of p38, ERK and JNK but not of Akt (Ser473) in the activated platelets. Images are representative of three separate experiments. The relative protein expression levels were quantified by Quantity One software. Data represent the mean ± SEM of at least three independent experiments performed in triplicate. * p

    Techniques Used: Incubation, Expressing, Software

    13) Product Images from "Endoplasmic Reticulum Stress Is a Danger Signal Promoting Innate Inflammatory Responses in Bronchial Epithelial Cells"

    Article Title: Endoplasmic Reticulum Stress Is a Danger Signal Promoting Innate Inflammatory Responses in Bronchial Epithelial Cells

    Journal: Journal of Innate Immunity

    doi: 10.1159/000447668

    PERK modulates p38 and ERK activation. a BEAS-2B cells were transfected with siRNA against PERK. After 42 h, cells were pretreated with DMSO or thapsigargin (Thaps) for 1 h and subsequently stimulated with pIC for 5 min. b BEAS-2B cells were pretreated
    Figure Legend Snippet: PERK modulates p38 and ERK activation. a BEAS-2B cells were transfected with siRNA against PERK. After 42 h, cells were pretreated with DMSO or thapsigargin (Thaps) for 1 h and subsequently stimulated with pIC for 5 min. b BEAS-2B cells were pretreated

    Techniques Used: Activation Assay, Transfection

    ER stress, via PERK- and ATF6-mediated p38 and ERK activation, is able to boost TLR- and NFκB-mediated inflammation in airway epithelial cells: under homeostatic conditions, direct stimulation of bronchial epithelial cells through PAMP/TLR (LPS
    Figure Legend Snippet: ER stress, via PERK- and ATF6-mediated p38 and ERK activation, is able to boost TLR- and NFκB-mediated inflammation in airway epithelial cells: under homeostatic conditions, direct stimulation of bronchial epithelial cells through PAMP/TLR (LPS

    Techniques Used: Activation Assay

    p38 and ERK mediate increased reactivity of bronchial epithelial cells to LPS and pIC upon ER stress induction. BEAS-2B ( a , b ) and hpBrEpC cells ( c , d ) were pretreated with the inhibitors of p38 (SB203580, 10 µ M ), ERK (U0126, 10 µ M ), and
    Figure Legend Snippet: p38 and ERK mediate increased reactivity of bronchial epithelial cells to LPS and pIC upon ER stress induction. BEAS-2B ( a , b ) and hpBrEpC cells ( c , d ) were pretreated with the inhibitors of p38 (SB203580, 10 µ M ), ERK (U0126, 10 µ M ), and

    Techniques Used:

    UPR induction results in enhanced p38 and ERK activity upon LPS stimulation. BEAS-2B cells were pretreated with DMSO or thapsigargin (Thaps) for 1 h and then stimulated with LPS for 15 min. Protein levels of activated, phosphorylated (p)-p38 ( a ), p-ERK
    Figure Legend Snippet: UPR induction results in enhanced p38 and ERK activity upon LPS stimulation. BEAS-2B cells were pretreated with DMSO or thapsigargin (Thaps) for 1 h and then stimulated with LPS for 15 min. Protein levels of activated, phosphorylated (p)-p38 ( a ), p-ERK

    Techniques Used: Activity Assay

    14) Product Images from "METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling"

    Article Title: METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21010199

    Effect of METTL3 knockdown on osteoblast proinflammatory cytokine production and MAPK and NF-κB signaling activation. ( A ) MC3T3-E1 cells were transfected with siRNA (METTL3 or negative control) and then cultured in osteogenic medium with or without 1 μg/mL LPS for 60 h. The expression of IL-6 , IL-12 , and TNF-α was examined by qRT-PCR. Gapdh was used as an internal control. ( B , C ) Cells transfected with siRNA (METTL3 or negative control) were cultured in osteogenic medium for 60 h and then treated with 1 μg/mL LPS for 0–120 min. MAPK and NF-κB phosphorylation was examined by western blotting, and VINCULIN was used as an internal reference. ( D ) Cells transfected with siRNA (METTL3 or negative control) were treated with the NF-κB inhibitor BAY 11-7082, the p38 inhibitor SB203580, the ERK inhibitor U0126, or the JNK inhibitor SP600125 for 1 h and then cultured in LPS-induced osteogenic medium for 60 h. The mRNA levels of IL-6 and TNF-α were detected by qRT-PCR. Gapdh was used as an internal control. The results are shown as the mean ± SD ( n = 3). * p
    Figure Legend Snippet: Effect of METTL3 knockdown on osteoblast proinflammatory cytokine production and MAPK and NF-κB signaling activation. ( A ) MC3T3-E1 cells were transfected with siRNA (METTL3 or negative control) and then cultured in osteogenic medium with or without 1 μg/mL LPS for 60 h. The expression of IL-6 , IL-12 , and TNF-α was examined by qRT-PCR. Gapdh was used as an internal control. ( B , C ) Cells transfected with siRNA (METTL3 or negative control) were cultured in osteogenic medium for 60 h and then treated with 1 μg/mL LPS for 0–120 min. MAPK and NF-κB phosphorylation was examined by western blotting, and VINCULIN was used as an internal reference. ( D ) Cells transfected with siRNA (METTL3 or negative control) were treated with the NF-κB inhibitor BAY 11-7082, the p38 inhibitor SB203580, the ERK inhibitor U0126, or the JNK inhibitor SP600125 for 1 h and then cultured in LPS-induced osteogenic medium for 60 h. The mRNA levels of IL-6 and TNF-α were detected by qRT-PCR. Gapdh was used as an internal control. The results are shown as the mean ± SD ( n = 3). * p

    Techniques Used: Activation Assay, Transfection, Negative Control, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot

    15) Product Images from "Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12"

    Article Title: Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113592

    Western blot and cell-based ELISA show that CXCL12 treatment of transfected CHO cells differentially activates the ERK and AKT pathways. Serum starved ( A ) CHO-CXCR4, ( B ) CHO-ACKR3 and ( C ) CHO-CXCR4-ACKR3 cells were stimulated with 10 nM CXCL12 for 5, 15 and 120 min. ( Top ) Cells were lysed and immunoblots probed with p-ERK or p-Akt, stripped and re-probed for pan-ERK or pan-AKT and GAPDH as a loading control. Images are representative of three independent experiments. ( Bottom ) Cells were fixed with methanol and cell-based ELISA was performed as per protocol using p-ERK and total-ERK antibodies with fluorescence intensity readings at 600 and 450 nm. Data represents the mean ± SEM of three independent experiments and statistical significance was calculated using a one way ANOVA (* p
    Figure Legend Snippet: Western blot and cell-based ELISA show that CXCL12 treatment of transfected CHO cells differentially activates the ERK and AKT pathways. Serum starved ( A ) CHO-CXCR4, ( B ) CHO-ACKR3 and ( C ) CHO-CXCR4-ACKR3 cells were stimulated with 10 nM CXCL12 for 5, 15 and 120 min. ( Top ) Cells were lysed and immunoblots probed with p-ERK or p-Akt, stripped and re-probed for pan-ERK or pan-AKT and GAPDH as a loading control. Images are representative of three independent experiments. ( Bottom ) Cells were fixed with methanol and cell-based ELISA was performed as per protocol using p-ERK and total-ERK antibodies with fluorescence intensity readings at 600 and 450 nm. Data represents the mean ± SEM of three independent experiments and statistical significance was calculated using a one way ANOVA (* p

    Techniques Used: Western Blot, In-Cell ELISA, Transfection, Fluorescence

    16) Product Images from "Organic Selenium Ameliorates Staphylococcus aureus-Induced Mastitis in Rats by Inhibiting the Activation of NF-κB and MAPK Signaling Pathways"

    Article Title: Organic Selenium Ameliorates Staphylococcus aureus-Induced Mastitis in Rats by Inhibiting the Activation of NF-κB and MAPK Signaling Pathways

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2020.00443

    The effects of OS on MAPKs signaling pathway in the mammary glands. Western blot was used to assay the phosphorylation levels of p38, JNK, and ERK proteins. Data are mean ± SEM ( n ≥ 3). ## p
    Figure Legend Snippet: The effects of OS on MAPKs signaling pathway in the mammary glands. Western blot was used to assay the phosphorylation levels of p38, JNK, and ERK proteins. Data are mean ± SEM ( n ≥ 3). ## p

    Techniques Used: Western Blot

    17) Product Images from "Isoform specific FBXW7 mediates NOTCH1 Abruptex mutation C1133Y deregulation in oral squamous cell carcinoma"

    Article Title: Isoform specific FBXW7 mediates NOTCH1 Abruptex mutation C1133Y deregulation in oral squamous cell carcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-02873-4

    FBXW7β is critical for the activation of AKT/ERK/NFκB pathway prompted by NOTCH1 C1133Y mutation in OSCC cells. a , b AKT/ERK/NFκB signaling activities were evaluated by western blot analysis in HN6 ( a ) and CAL27 cells ( b ). The gray values of images demonstrated that FBXW7β transfection decreased phosphorylation of AKT/ERK/NFκB and reversed the NOTCH1 C1133Y -induced activation of AKT/ERK/NFκB phosphorylation. c , d Overexpression of FBXW7β reversed the increased protein expression levels of EMT markers and prevented the decrease in E-cadherin and β-Catenin caused by NOTCH1 C1133Y in HN6 ( c ) and CAL27 ( d ) cells. e The expression of p-AKT, p-ERK, and p-NFκB in xenografted mice was ascertained using IHC assay on tumor sections. The percentages of positive cells were acquired from three separate images and the qualification was presented. Scale bar, 20 μm. All the results were shown as mean ± SD. * p
    Figure Legend Snippet: FBXW7β is critical for the activation of AKT/ERK/NFκB pathway prompted by NOTCH1 C1133Y mutation in OSCC cells. a , b AKT/ERK/NFκB signaling activities were evaluated by western blot analysis in HN6 ( a ) and CAL27 cells ( b ). The gray values of images demonstrated that FBXW7β transfection decreased phosphorylation of AKT/ERK/NFκB and reversed the NOTCH1 C1133Y -induced activation of AKT/ERK/NFκB phosphorylation. c , d Overexpression of FBXW7β reversed the increased protein expression levels of EMT markers and prevented the decrease in E-cadherin and β-Catenin caused by NOTCH1 C1133Y in HN6 ( c ) and CAL27 ( d ) cells. e The expression of p-AKT, p-ERK, and p-NFκB in xenografted mice was ascertained using IHC assay on tumor sections. The percentages of positive cells were acquired from three separate images and the qualification was presented. Scale bar, 20 μm. All the results were shown as mean ± SD. * p

    Techniques Used: Activation Assay, Mutagenesis, Western Blot, Transfection, Over Expression, Expressing, Mouse Assay, Immunohistochemistry

    18) Product Images from "Activation of AKT signaling promotes cell growth and survival in ?7?1 integrin-mediated alleviation of muscular dystrophy"

    Article Title: Activation of AKT signaling promotes cell growth and survival in ?7?1 integrin-mediated alleviation of muscular dystrophy

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbadis.2011.01.002

    Phosphorylation of BAD and ERK is increased and apoptosis is inhibited in α7BX2- mdx/utr −/− mice. (A) BAD phosphorylation (Ser112) was examined in 5 week-old wild type, mdx, mdx/utr −/− , and α7BX2- mdx/utr
    Figure Legend Snippet: Phosphorylation of BAD and ERK is increased and apoptosis is inhibited in α7BX2- mdx/utr −/− mice. (A) BAD phosphorylation (Ser112) was examined in 5 week-old wild type, mdx, mdx/utr −/− , and α7BX2- mdx/utr

    Techniques Used: Mouse Assay

    19) Product Images from "Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients with severe asthma"

    Article Title: Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients with severe asthma

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2012.07.017

    Comparison of induced MAPKs in ASMCs of nonasthmatic subjects and patients with nonsevere and severe asthma. ASMCs were stimulated with TNF-α (10 ng/mL) for 15 minutes. Phosphorylated and total p38 ( A ), JNK ( B ), and ERK ( C ) levels were assessed
    Figure Legend Snippet: Comparison of induced MAPKs in ASMCs of nonasthmatic subjects and patients with nonsevere and severe asthma. ASMCs were stimulated with TNF-α (10 ng/mL) for 15 minutes. Phosphorylated and total p38 ( A ), JNK ( B ), and ERK ( C ) levels were assessed

    Techniques Used:

    20) Product Images from "IGFBP-1 hyperphosphorylation in response to leucine deprivation is mediated by the AAR pathway"

    Article Title: IGFBP-1 hyperphosphorylation in response to leucine deprivation is mediated by the AAR pathway

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2015.04.031

    Effects of ERK and/or GCN2 silencing on IGFBP-1 secretion and phosphorylation
    Figure Legend Snippet: Effects of ERK and/or GCN2 silencing on IGFBP-1 secretion and phosphorylation

    Techniques Used:

    Effects of ERK siRNA on IGFBP-1 secretion and phosphorylation
    Figure Legend Snippet: Effects of ERK siRNA on IGFBP-1 secretion and phosphorylation

    Techniques Used:

    21) Product Images from "Oestrogen‐activated autophagy has a negative effect on the anti‐osteoclastogenic function of oestrogen. Oestrogen‐activated autophagy has a negative effect on the anti‐osteoclastogenic function of oestrogen"

    Article Title: Oestrogen‐activated autophagy has a negative effect on the anti‐osteoclastogenic function of oestrogen. Oestrogen‐activated autophagy has a negative effect on the anti‐osteoclastogenic function of oestrogen

    Journal: Cell Proliferation

    doi: 10.1111/cpr.12789

    Treatment with 17β‐estradiol suppresses the TRAF6‐MAPK signalling pathway downstream of RANKL. A and B, OCPs were treated with or without RANKL for the indicated times. TRAF6, p‐ERK, p‐JNK and p‐P38 were detected using Western blot analyses. The histograms represent the comparisons of the expression levels of each protein between the RANKL‐lacking group and the RANKL group at the indicated time points. The expression of phosphorylated proteins is represented by the ratio of phosphorylated protein to total protein. C, OCPs were treated with or without RANKL for 2 h. Cell lysates were extracted for Co‐IP with anti‐RANK or anti‐TRAF6 antibody, and subsequently, precipitates were detected using Western blots with anti‐TRAF6 or anti‐RANK antibody, respectively. Data are expressed as the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: Treatment with 17β‐estradiol suppresses the TRAF6‐MAPK signalling pathway downstream of RANKL. A and B, OCPs were treated with or without RANKL for the indicated times. TRAF6, p‐ERK, p‐JNK and p‐P38 were detected using Western blot analyses. The histograms represent the comparisons of the expression levels of each protein between the RANKL‐lacking group and the RANKL group at the indicated time points. The expression of phosphorylated proteins is represented by the ratio of phosphorylated protein to total protein. C, OCPs were treated with or without RANKL for 2 h. Cell lysates were extracted for Co‐IP with anti‐RANK or anti‐TRAF6 antibody, and subsequently, precipitates were detected using Western blots with anti‐TRAF6 or anti‐RANK antibody, respectively. Data are expressed as the mean ± SEM from three independent experiments. * P

    Techniques Used: Western Blot, Expressing, Co-Immunoprecipitation Assay

    22) Product Images from "PEITC triggers multiple forms of cell death by GSH-iron-ROS regulation in K7M2 murine osteosarcoma cells"

    Article Title: PEITC triggers multiple forms of cell death by GSH-iron-ROS regulation in K7M2 murine osteosarcoma cells

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/s41401-020-0376-8

    Immunohistochemical analysis of tumor tissue from osteosarcoma mice treated by PEITC. a Immunohistochemical staining analysis of Ki67 (a marker of proliferation), C-caspase3 (a marker of apoptosis), LC3B (a marker of autophagy), and GPx4 (a marker of ferroptosis) in tumor tissues. b , c Protein expression levels and quantitative analysis of Cdc2, GPx4, C-caspase3, and LC3B in tumor tissues. d , e Protein expression levels and quantitative analysis of TfR1, DMT1, FPN, and FTH1 in tumor tissues. f , g Phosphorylation levels and quantitative analysis of ERK, p38, and JNK in tumor tissues. All data were presented as mean ± SD, n = 3. * P
    Figure Legend Snippet: Immunohistochemical analysis of tumor tissue from osteosarcoma mice treated by PEITC. a Immunohistochemical staining analysis of Ki67 (a marker of proliferation), C-caspase3 (a marker of apoptosis), LC3B (a marker of autophagy), and GPx4 (a marker of ferroptosis) in tumor tissues. b , c Protein expression levels and quantitative analysis of Cdc2, GPx4, C-caspase3, and LC3B in tumor tissues. d , e Protein expression levels and quantitative analysis of TfR1, DMT1, FPN, and FTH1 in tumor tissues. f , g Phosphorylation levels and quantitative analysis of ERK, p38, and JNK in tumor tissues. All data were presented as mean ± SD, n = 3. * P

    Techniques Used: Immunohistochemistry, Mouse Assay, Staining, Marker, Expressing

    PEITC induced cell death via the generation of ROS in K7M2 osteosarcoma cell. a , b Colony formation assay of K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. c EdU staining assay of the proliferation ability of K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. d , e Protein expression levels and quantitative analysis of TfR1, DMT1, FTH1, and FPN in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. f , g Protein expression levels and quantitative analysis of LC3B, GPx4, C-caspase3, Bcl-2, and Cdc2 in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. h , i Phosphorylation levels and quantitative analysis of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. All data were presented as mean ± SD, n = 3. * P
    Figure Legend Snippet: PEITC induced cell death via the generation of ROS in K7M2 osteosarcoma cell. a , b Colony formation assay of K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. c EdU staining assay of the proliferation ability of K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. d , e Protein expression levels and quantitative analysis of TfR1, DMT1, FTH1, and FPN in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. f , g Protein expression levels and quantitative analysis of LC3B, GPx4, C-caspase3, Bcl-2, and Cdc2 in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. h , i Phosphorylation levels and quantitative analysis of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by 30 μM PEITC in the presence of NAC or not for 24 h. All data were presented as mean ± SD, n = 3. * P

    Techniques Used: Colony Assay, Staining, Expressing

    PEITC activated MAPK signaling pathway in K7M2 osteosarcoma cells. a , b Phosphorylation levels of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4, 12, 24, and 48 h. c , d Quantitative analysis of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4, 12, 24, and 48 h. All data were presented as mean ± SD, n = 3. * P
    Figure Legend Snippet: PEITC activated MAPK signaling pathway in K7M2 osteosarcoma cells. a , b Phosphorylation levels of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4, 12, 24, and 48 h. c , d Quantitative analysis of ERK, p38, and JNK in K7M2 osteosarcoma cells treated by indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4, 12, 24, and 48 h. All data were presented as mean ± SD, n = 3. * P

    Techniques Used:

    23) Product Images from "Fibroblast-Like-Synoviocytes Mediate Secretion of Pro-Inflammatory Cytokines via ERK and JNK MAPKs in Ti-Particle-Induced Osteolysis"

    Article Title: Fibroblast-Like-Synoviocytes Mediate Secretion of Pro-Inflammatory Cytokines via ERK and JNK MAPKs in Ti-Particle-Induced Osteolysis

    Journal: Materials

    doi: 10.3390/ma13163628

    Effect of Ti particle on the activation of MAPKs in SW982 cells. ( A ( a )) Representative Western blots showing protein levels of total and phosphorylated forms of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinases (MAPKs) after treatment with Ti particles (cell-to-particle ratio of 1:100) of SW982 cells for 15, 30, 60, and 120 min. Here, β-actin was taken as control. ( A ( b )) Fusion FX software was utilized for quantitative densitometric analysis of the proteins. ( B ) AP1-luc reporter plasmids were transfected to SW982 cells for 24 h and luciferase activity was analyzed. Renilla luciferase activity was used for normalization. The results are demonstrated as means ± SDs of three independent experiments. In the graphical representations, * p
    Figure Legend Snippet: Effect of Ti particle on the activation of MAPKs in SW982 cells. ( A ( a )) Representative Western blots showing protein levels of total and phosphorylated forms of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinases (MAPKs) after treatment with Ti particles (cell-to-particle ratio of 1:100) of SW982 cells for 15, 30, 60, and 120 min. Here, β-actin was taken as control. ( A ( b )) Fusion FX software was utilized for quantitative densitometric analysis of the proteins. ( B ) AP1-luc reporter plasmids were transfected to SW982 cells for 24 h and luciferase activity was analyzed. Renilla luciferase activity was used for normalization. The results are demonstrated as means ± SDs of three independent experiments. In the graphical representations, * p

    Techniques Used: Activation Assay, Western Blot, Software, Transfection, Luciferase, Activity Assay

    Co-inhibition of ERK and JNK MAPKs suppresses the expression of pro-inflammatory cytokines in SW982 cell line. Ti particles were used to treat (Cell-to-particle ratio of 1:100) SW982 cells prior to incubation (30 min) with either PD98059 (5 µM), SP600125 (5 µM), or PD98059 along with SP600125 for 24 h. RT-PCR analysis showing the mRNA expression of ( A(a) ) IL-6, ( A(b) ) IL-1β, and ( A(c) ) TNF α after co-inhibition of ERK and JNK MAPKs. The mRNA expression of each targeted gene was normalized to GAPDH. ( B ) ELISA results demonstrated secretion levels of ( B(a) ) IL-6, ( B(b) ) IL-1β, and ( B(c) ) TNF α after co-inhibition of ERK and JNK MAPKs. The results are presented as means ± SDs of three independent experiments. In the graphical representations, ** p
    Figure Legend Snippet: Co-inhibition of ERK and JNK MAPKs suppresses the expression of pro-inflammatory cytokines in SW982 cell line. Ti particles were used to treat (Cell-to-particle ratio of 1:100) SW982 cells prior to incubation (30 min) with either PD98059 (5 µM), SP600125 (5 µM), or PD98059 along with SP600125 for 24 h. RT-PCR analysis showing the mRNA expression of ( A(a) ) IL-6, ( A(b) ) IL-1β, and ( A(c) ) TNF α after co-inhibition of ERK and JNK MAPKs. The mRNA expression of each targeted gene was normalized to GAPDH. ( B ) ELISA results demonstrated secretion levels of ( B(a) ) IL-6, ( B(b) ) IL-1β, and ( B(c) ) TNF α after co-inhibition of ERK and JNK MAPKs. The results are presented as means ± SDs of three independent experiments. In the graphical representations, ** p

    Techniques Used: Inhibition, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    24) Product Images from "SHIP2 inhibition alters redox‐induced PI3K/AKT and MAP kinase pathways via PTEN over‐activation in cervical cancer cells"

    Article Title: SHIP2 inhibition alters redox‐induced PI3K/AKT and MAP kinase pathways via PTEN over‐activation in cervical cancer cells

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12967

    SHIP inhibition alters PI(3,4,5)P3 levels on H 2 O 2 stimulation. (A) HeLa cells were first transfected with the plasmid GFP‐tagged BTK–PH domain, and 6 h later they were treated with vehicle or AS19 for 24 h. Subsequently, cells were treated with 1 m m H 2 O 2 for 1 h, then fixed and mounted. Fluorescent images representing PI(3,4,5)P3 levels are shown. Cells pretreated with 200 n m wortmannin (Wort) were used as a control. Arrows indicate PI(3,4,5)P3 signal. Upper and lower panels are from two biological replicates for each condition. Scale bars: 10 μ m . (B) Kinetics of AKT and ERK phosphorylation in HeLa cells after 24 h of SHIP2 inhibition and subsequent H 2 O 2 treatment (1 m m ). Lysate collected and analyzed for the indicated times. Short and long exposure for phosphorylated (p)‐AKT and p‐ERK were shown. (C) HeLa cells were treated with a vehicle or AS19 for 24 h, followed by AKT activator (SC79), 20 µ m for 2 h, then 1 m m H 2 O 2 , and then collected at the indicated times. Short and long exposure of the blots were shown. (D) SHIP2 inhibition activates PTEN upon increase in ROS. HeLa cells were treated with vehicle or AS19 for 24 h, then followed by 1 m m H 2 O 2 for 4 h. Lysates were analyzed for the indicated antibodies. H 2 O 2 treatment was performed in three biological replicates for each condition. (E) Bar graph summarizing PTEN phosphorylation levels in HeLa cells for each treatment. Data are the means ± SEM from three independent experiments. One‐way ANOVA, F (3, 8) = 11.91, P = 0.0025. Tukey's multiple comparisons test, ** P ˂ 0.01. C‐, vehicle‐treated cells.
    Figure Legend Snippet: SHIP inhibition alters PI(3,4,5)P3 levels on H 2 O 2 stimulation. (A) HeLa cells were first transfected with the plasmid GFP‐tagged BTK–PH domain, and 6 h later they were treated with vehicle or AS19 for 24 h. Subsequently, cells were treated with 1 m m H 2 O 2 for 1 h, then fixed and mounted. Fluorescent images representing PI(3,4,5)P3 levels are shown. Cells pretreated with 200 n m wortmannin (Wort) were used as a control. Arrows indicate PI(3,4,5)P3 signal. Upper and lower panels are from two biological replicates for each condition. Scale bars: 10 μ m . (B) Kinetics of AKT and ERK phosphorylation in HeLa cells after 24 h of SHIP2 inhibition and subsequent H 2 O 2 treatment (1 m m ). Lysate collected and analyzed for the indicated times. Short and long exposure for phosphorylated (p)‐AKT and p‐ERK were shown. (C) HeLa cells were treated with a vehicle or AS19 for 24 h, followed by AKT activator (SC79), 20 µ m for 2 h, then 1 m m H 2 O 2 , and then collected at the indicated times. Short and long exposure of the blots were shown. (D) SHIP2 inhibition activates PTEN upon increase in ROS. HeLa cells were treated with vehicle or AS19 for 24 h, then followed by 1 m m H 2 O 2 for 4 h. Lysates were analyzed for the indicated antibodies. H 2 O 2 treatment was performed in three biological replicates for each condition. (E) Bar graph summarizing PTEN phosphorylation levels in HeLa cells for each treatment. Data are the means ± SEM from three independent experiments. One‐way ANOVA, F (3, 8) = 11.91, P = 0.0025. Tukey's multiple comparisons test, ** P ˂ 0.01. C‐, vehicle‐treated cells.

    Techniques Used: Inhibition, Transfection, Plasmid Preparation

    Catalytic inhibition of PTEN rescues PI(3,4)P2 levels and ERK phosphorylation. (A) HeLa cells were first transfected with GFP‐tagged TAPP1‐PH domain plasmid; 6 h later they were treated overnight with vehicle or AS19 followed by 1 m m H 2 O 2 for 1 h. Cells were then fixed and mounted. Fluorescent images of PI(3,4)P2 levels are shown. Cells pretreated with 2 µ m PTEN inhibitor (VO‐OHpic) were used as a control. Arrows indicate PI(3,4)P2 signal. Scale bars: 10 μ m . (B) PTEN inhibition rescues MAPK activation. HeLa cells were treated with vehicle or AS19 for 24 h, followed by 2 µ m PTEN inhibitor for 2 h, then 1 m m H 2 O 2 for 4 h. Cell lysates were analyzed for the indicated antibodies. (C) Bar graph summarizing ERK and AKT phosphorylation levels for each treatment. Data are the means ± SEM from three independent experiments, each in duplicates. p‐ERK/ERK, one‐way ANOVA, F (5, 12) = 101.1, P
    Figure Legend Snippet: Catalytic inhibition of PTEN rescues PI(3,4)P2 levels and ERK phosphorylation. (A) HeLa cells were first transfected with GFP‐tagged TAPP1‐PH domain plasmid; 6 h later they were treated overnight with vehicle or AS19 followed by 1 m m H 2 O 2 for 1 h. Cells were then fixed and mounted. Fluorescent images of PI(3,4)P2 levels are shown. Cells pretreated with 2 µ m PTEN inhibitor (VO‐OHpic) were used as a control. Arrows indicate PI(3,4)P2 signal. Scale bars: 10 μ m . (B) PTEN inhibition rescues MAPK activation. HeLa cells were treated with vehicle or AS19 for 24 h, followed by 2 µ m PTEN inhibitor for 2 h, then 1 m m H 2 O 2 for 4 h. Cell lysates were analyzed for the indicated antibodies. (C) Bar graph summarizing ERK and AKT phosphorylation levels for each treatment. Data are the means ± SEM from three independent experiments, each in duplicates. p‐ERK/ERK, one‐way ANOVA, F (5, 12) = 101.1, P

    Techniques Used: Inhibition, Transfection, Plasmid Preparation, Activation Assay

    25) Product Images from "The Inhibition of RANKL-Induced Osteoclastogenesis through the Suppression of p38 Signaling Pathway by Naringenin and Attenuation of Titanium-Particle-Induced Osteolysis"

    Article Title: The Inhibition of RANKL-Induced Osteoclastogenesis through the Suppression of p38 Signaling Pathway by Naringenin and Attenuation of Titanium-Particle-Induced Osteolysis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms151221913

    Naringenin (NAR)-mediated effects on receptor activator of the nuclear factor-κB ligand RANKL-induced nuclear factor κB (NF-κB), c-Jun N -terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), signaling ( A ) The levels of p-IκBα, IκBα, p-JNK and p-ERK were unaffected by exposure to 200 μM NAR; ( B ) Luciferase reporter assays showed that NAR did not affect RANKL-induced NF-κB signaling.
    Figure Legend Snippet: Naringenin (NAR)-mediated effects on receptor activator of the nuclear factor-κB ligand RANKL-induced nuclear factor κB (NF-κB), c-Jun N -terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), signaling ( A ) The levels of p-IκBα, IκBα, p-JNK and p-ERK were unaffected by exposure to 200 μM NAR; ( B ) Luciferase reporter assays showed that NAR did not affect RANKL-induced NF-κB signaling.

    Techniques Used: Luciferase

    26) Product Images from "Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation"

    Article Title: Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2015/682019

    Effects of O. japonicus on the LPS-induced phosphorylation of JNK, p38 MAPK, and ERK in THP-1 cells. Cells were pretreated for 20 h with various concentrations of O. japonicus (10 or 25 μ g/mL) before exposure to LPS (1 μ g/mL) for 4 h, and JNK, p38 MAPK, and ERK protein levels were determined by immunoblotting. Densitometric analyses are presented as the relative ratios of JNK (a) or p-JNK (d), p38 MAPK (b) or p-p38 MAPK (e), and ERK (c) or p-ERK (f) to β -actin. The data represent the means ± SD of three independent samples. * P
    Figure Legend Snippet: Effects of O. japonicus on the LPS-induced phosphorylation of JNK, p38 MAPK, and ERK in THP-1 cells. Cells were pretreated for 20 h with various concentrations of O. japonicus (10 or 25 μ g/mL) before exposure to LPS (1 μ g/mL) for 4 h, and JNK, p38 MAPK, and ERK protein levels were determined by immunoblotting. Densitometric analyses are presented as the relative ratios of JNK (a) or p-JNK (d), p38 MAPK (b) or p-p38 MAPK (e), and ERK (c) or p-ERK (f) to β -actin. The data represent the means ± SD of three independent samples. * P

    Techniques Used:

    27) Product Images from "Gab1 is essential for membrane translocation, activity and integrity of mTORCs after EGF stimulation in urothelial cell carcinoma"

    Article Title: Gab1 is essential for membrane translocation, activity and integrity of mTORCs after EGF stimulation in urothelial cell carcinoma

    Journal: Oncotarget

    doi:

    Gab1 is important for the activation of mTORC signaling after EGF stimulation (A) T24 cells were transfected with Gab1 siRNA, treated with LY294002 (20 μM) for 60 min, stimulated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204 and Gab1. (B) E6 cells transfected with Myc-Gab1wt were serum-starved, and the treatment was performed in a similar manner to that described in A . Immunoblotting was performed to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204, Gab1 and myc. (C) E6 cells were transfected with Myc-Gab1wt and Myc-Gab1ΔPH, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473 and Gab1. (D) T24 cells were transfected with raptor siRNA, rictor siRNA and Myc-Gab1wt, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, raptor, rictor, p70S6K-pT389, AKT-pS473, Gab1 and myc. For all experiments, n = 3.
    Figure Legend Snippet: Gab1 is important for the activation of mTORC signaling after EGF stimulation (A) T24 cells were transfected with Gab1 siRNA, treated with LY294002 (20 μM) for 60 min, stimulated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204 and Gab1. (B) E6 cells transfected with Myc-Gab1wt were serum-starved, and the treatment was performed in a similar manner to that described in A . Immunoblotting was performed to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473, ERK-pT202/Y204, Gab1 and myc. (C) E6 cells were transfected with Myc-Gab1wt and Myc-Gab1ΔPH, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, p70S6K-pT389, AKT-pS473 and Gab1. (D) T24 cells were transfected with raptor siRNA, rictor siRNA and Myc-Gab1wt, treated with 100 ng/ml EGF for 10 min and then immunoblotted to detect mTOR-pS2448, mTOR-pS2481, raptor, rictor, p70S6K-pT389, AKT-pS473, Gab1 and myc. For all experiments, n = 3.

    Techniques Used: Activation Assay, Transfection

    28) Product Images from "Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium"

    Article Title: Caveolin and MAP kinase interaction in angiotensin II preconditioning of the myocardium

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2007.00067.x

    Distribution of p38MAPKα, p38MAPKβ, ERK and JNK in different fraction of the Ang II preconditioned heart.
    Figure Legend Snippet: Distribution of p38MAPKα, p38MAPKβ, ERK and JNK in different fraction of the Ang II preconditioned heart.

    Techniques Used:

    Differential interaction of p38MAPK, JNK, ERK 1/2 and eNOS with caveolin-1 and caveolin-3 during ischaemia reperfusion, Ang II precondition in presence or absence of NAC or apocynin.The results are mean ± SEM of six animal per group * P
    Figure Legend Snippet: Differential interaction of p38MAPK, JNK, ERK 1/2 and eNOS with caveolin-1 and caveolin-3 during ischaemia reperfusion, Ang II precondition in presence or absence of NAC or apocynin.The results are mean ± SEM of six animal per group * P

    Techniques Used:

    29) Product Images from "Effect of a long-term treatment with a low-dose granulocyte colony-stimulating factor on post-infarction process in the heart"

    Article Title: Effect of a long-term treatment with a low-dose granulocyte colony-stimulating factor on post-infarction process in the heart

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00294.x

    Expression of G-CSFR and its downstream signalling in hearts and its modulation by G-CSF. ( A ) Western and immunohistochemical analyses of G-CSFR expression. Immunohistochemistry shows localization of G-CSFRs on cardiomyocytes, interstitial cells and endothelial cells. Arrows in the right panel indicate G-CSFR-positive endothelial cells and interstitial cells. ( B ) Western analysis of the expression of STAT3, Akt, ERK and their phosphorylated forms (p-STAT3, p-Akt and p-ERK). Immunohistochemistry shows the distribution of p-STAT3 and p-Akt in the myocardium. Bars, 100 μm. n = 4 each for the saline-treated sham, the G-CSF-treated sham and the saline-treated MI groups; n = 5 for the G-CSF-treated MI group. * P
    Figure Legend Snippet: Expression of G-CSFR and its downstream signalling in hearts and its modulation by G-CSF. ( A ) Western and immunohistochemical analyses of G-CSFR expression. Immunohistochemistry shows localization of G-CSFRs on cardiomyocytes, interstitial cells and endothelial cells. Arrows in the right panel indicate G-CSFR-positive endothelial cells and interstitial cells. ( B ) Western analysis of the expression of STAT3, Akt, ERK and their phosphorylated forms (p-STAT3, p-Akt and p-ERK). Immunohistochemistry shows the distribution of p-STAT3 and p-Akt in the myocardium. Bars, 100 μm. n = 4 each for the saline-treated sham, the G-CSF-treated sham and the saline-treated MI groups; n = 5 for the G-CSF-treated MI group. * P

    Techniques Used: Expressing, Western Blot, Immunohistochemistry

    30) Product Images from "Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR"

    Article Title: Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2014.164

    The mutation in EGFR reduces signal transduction and cell proliferation ( a ) Western blotting was performed for p-EGFR, EGFR, p-ERK, ERK, p-Akt and Akt on lysates from untransfected (U), wild-type EGFR (WT) or mutant EGFR (MUT) transfected MCF-7 cells after EGF stimulation for the indicated times. MCF-7 cells express very low endogenous EGFR and therefore EGFR is undetectable in UT cells. Arrows indicate phospho and total EGFR species in top and second row blots respectively. Note the higher molecular weight species of EGFR in the WT samples in the second row blot are phosphorylated receptor and directly correlate with phosphorylated EGFR as detected in the top row blot. ( b ) Cell proliferation was quantified in CHO-K1 cells transfected with GFP, wild-type EGFR-GFP or mutant EGFR-GFP in starved (−), normal growth (10% fetal calf serum) or EGF-stimulated (EGF) conditions. ( c ) GFP-positive cells were counted and normalized against total cell number, and the cell growth rate was then calculated from these data.
    Figure Legend Snippet: The mutation in EGFR reduces signal transduction and cell proliferation ( a ) Western blotting was performed for p-EGFR, EGFR, p-ERK, ERK, p-Akt and Akt on lysates from untransfected (U), wild-type EGFR (WT) or mutant EGFR (MUT) transfected MCF-7 cells after EGF stimulation for the indicated times. MCF-7 cells express very low endogenous EGFR and therefore EGFR is undetectable in UT cells. Arrows indicate phospho and total EGFR species in top and second row blots respectively. Note the higher molecular weight species of EGFR in the WT samples in the second row blot are phosphorylated receptor and directly correlate with phosphorylated EGFR as detected in the top row blot. ( b ) Cell proliferation was quantified in CHO-K1 cells transfected with GFP, wild-type EGFR-GFP or mutant EGFR-GFP in starved (−), normal growth (10% fetal calf serum) or EGF-stimulated (EGF) conditions. ( c ) GFP-positive cells were counted and normalized against total cell number, and the cell growth rate was then calculated from these data.

    Techniques Used: Mutagenesis, Transduction, Western Blot, Transfection, Molecular Weight

    31) Product Images from "Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer"

    Article Title: Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125515

    miR-193b regulates the expression of KRAS by directly targeting its 3′-UTR. (A) The TargetScan online database ( http://www.targetscan.org ) predicted KRAS as an miR-193b target. The site between 1074 and 1080 (boxed region), one of the two predicted miR-193b binding sites in the 3′-UTR of KRAS, is shown, highlighting the evolutionarily conservation among humans (hsa), the chimpanzees (Ptr), rhesus monkey (Mml), bushbaby (Oga), and treeshrew (Tbe). (B) The two miR-193b binding sites at positions 303 to 309 and 1074–1080 in the KRAS 3′-UTR, identified using the TargetScan online database ( http://www.targetscan.org ), are shown. Seed sequences were mutated as shown in the boxed regions. The wild-type or mutant constructs were inserted into the pGL3 vector directly downstream of the luciferase gene. (C) 293A cells were co-transfected with miR-193b or scrambled oligonucleotide, wild-type or mutant firefly luciferase constructs of the KRAS 3′-UTR segment containing miR-193b binding sites, and Renilla luciferase (endogenous control). Luciferase activity was measured 24 hours after transfection using the Dual-Luciferase Reporter Assay Systems. Renilla-normalized luciferase activity is expressed relative to that obtained for the scrambled oligonucleotide under each condition. WT, wild-type; Mut, mutant. (D) KRAS and CCND1 protein levels were assessed by Western blotting 48 hours after transfection. (E) The protein levels of p-ERK and p-Akt were evaluated by Western blotting 48 hours after miR-193b transfection.
    Figure Legend Snippet: miR-193b regulates the expression of KRAS by directly targeting its 3′-UTR. (A) The TargetScan online database ( http://www.targetscan.org ) predicted KRAS as an miR-193b target. The site between 1074 and 1080 (boxed region), one of the two predicted miR-193b binding sites in the 3′-UTR of KRAS, is shown, highlighting the evolutionarily conservation among humans (hsa), the chimpanzees (Ptr), rhesus monkey (Mml), bushbaby (Oga), and treeshrew (Tbe). (B) The two miR-193b binding sites at positions 303 to 309 and 1074–1080 in the KRAS 3′-UTR, identified using the TargetScan online database ( http://www.targetscan.org ), are shown. Seed sequences were mutated as shown in the boxed regions. The wild-type or mutant constructs were inserted into the pGL3 vector directly downstream of the luciferase gene. (C) 293A cells were co-transfected with miR-193b or scrambled oligonucleotide, wild-type or mutant firefly luciferase constructs of the KRAS 3′-UTR segment containing miR-193b binding sites, and Renilla luciferase (endogenous control). Luciferase activity was measured 24 hours after transfection using the Dual-Luciferase Reporter Assay Systems. Renilla-normalized luciferase activity is expressed relative to that obtained for the scrambled oligonucleotide under each condition. WT, wild-type; Mut, mutant. (D) KRAS and CCND1 protein levels were assessed by Western blotting 48 hours after transfection. (E) The protein levels of p-ERK and p-Akt were evaluated by Western blotting 48 hours after miR-193b transfection.

    Techniques Used: Expressing, Binding Assay, Mutagenesis, Construct, Plasmid Preparation, Luciferase, Transfection, Activity Assay, Reporter Assay, Western Blot

    32) Product Images from "DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance"

    Article Title: DOK3 Negatively Regulates LPS Responses and Endotoxin Tolerance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039967

    LPS-induced degradation of DOK3 mediates ERK activation. (A) RAW264.7 macrophages, transduced with 23 nt plus hairpin shRNA designed to knock down DOK3 or a control luciferase knock down, were lysed and decreased DOK3 expression was confirmed by immunoblotting. Cells were and stimulated with 1 µg/ml LPS for the indicated time and cell lysates were immunoblotted for pERK, ERK, and GAPDH. (B) Wild type (WT) or DOK3-deficient BMM were stimulated with 1 µg/ml LPS and cell lysates were immunblotted for pERK, ERK, and the GAPDH. The amount of DOK3 and Grb2 was detected by immunoblot in cell lysates. (C) BMM cells were pretreated with DMSO or 10 µM MG132 and stimulated with 1 µg/ml LPS for the indicated times. Lysates were immunoblotted for phosphorylated ERK (pERK), ERK, and DOK3. (D) RAW264.7 macrophages transfected with FLAG-tagged mouse DOK3- were stimulated with 1 µg/ml LPS for the indicated time and cell lysates were immunoblotted for pERK, ERK, FLAG and GAPDH. Data are representative of three independent experiments.
    Figure Legend Snippet: LPS-induced degradation of DOK3 mediates ERK activation. (A) RAW264.7 macrophages, transduced with 23 nt plus hairpin shRNA designed to knock down DOK3 or a control luciferase knock down, were lysed and decreased DOK3 expression was confirmed by immunoblotting. Cells were and stimulated with 1 µg/ml LPS for the indicated time and cell lysates were immunoblotted for pERK, ERK, and GAPDH. (B) Wild type (WT) or DOK3-deficient BMM were stimulated with 1 µg/ml LPS and cell lysates were immunblotted for pERK, ERK, and the GAPDH. The amount of DOK3 and Grb2 was detected by immunoblot in cell lysates. (C) BMM cells were pretreated with DMSO or 10 µM MG132 and stimulated with 1 µg/ml LPS for the indicated times. Lysates were immunoblotted for phosphorylated ERK (pERK), ERK, and DOK3. (D) RAW264.7 macrophages transfected with FLAG-tagged mouse DOK3- were stimulated with 1 µg/ml LPS for the indicated time and cell lysates were immunoblotted for pERK, ERK, FLAG and GAPDH. Data are representative of three independent experiments.

    Techniques Used: Activation Assay, Transduction, shRNA, Luciferase, Expressing, Transfection

    Proposed model of DOK3 regulation of TLR4 signaling and endotoxin tolerance. In naïve cells, DOK3 associates with Grb2 and SOS1 constitutively. Upon LPS stimulation, DOK3 becomes ubiquitinated, possibly by Cbl-b, and degraded, thereby releasing Grb2. SOS1 is also degraded in a DOK3-dependent manner thus limiting ERK activation. In LPS-induced tolerant cells, DOK3 and SOS1 expression remains stable during repeated LPS challenge. DOK3 may limit ERK activation during tolerance by binding to and sequestering Grb2 and SOS1. Mediators of tolerance including IRAK-M, SHIP1, and SOCS1 are upregulated.
    Figure Legend Snippet: Proposed model of DOK3 regulation of TLR4 signaling and endotoxin tolerance. In naïve cells, DOK3 associates with Grb2 and SOS1 constitutively. Upon LPS stimulation, DOK3 becomes ubiquitinated, possibly by Cbl-b, and degraded, thereby releasing Grb2. SOS1 is also degraded in a DOK3-dependent manner thus limiting ERK activation. In LPS-induced tolerant cells, DOK3 and SOS1 expression remains stable during repeated LPS challenge. DOK3 may limit ERK activation during tolerance by binding to and sequestering Grb2 and SOS1. Mediators of tolerance including IRAK-M, SHIP1, and SOCS1 are upregulated.

    Techniques Used: Activation Assay, Expressing, Binding Assay

    DOK3 and SOS1 are stable during LPS-induced tolerance. (A) BMM were pretreated with 1 µg/ml LPS for 18 hours, rested in fresh media for 2 hours and re-stimulated along with naïve cells with 1 µg/ml LPS for indicated time and cell lysates were immunoblotted for DOK3, pERK, ERK, and GAPDH. (B) Tolerant BMM were stimulated with 1 µg/ml LPS and cell lysates were immunoprecipitated with anti-DOK3 antibody and immunoblotted for Grb2 and DOK3. (C) Tolerant wild type and DOK3-deficient BMM were re-stimulated with 1 µg/ml LPS and lysates immunoblotted for SOS1 and JNK (loading control). Data are representative of three independent experiments.
    Figure Legend Snippet: DOK3 and SOS1 are stable during LPS-induced tolerance. (A) BMM were pretreated with 1 µg/ml LPS for 18 hours, rested in fresh media for 2 hours and re-stimulated along with naïve cells with 1 µg/ml LPS for indicated time and cell lysates were immunoblotted for DOK3, pERK, ERK, and GAPDH. (B) Tolerant BMM were stimulated with 1 µg/ml LPS and cell lysates were immunoprecipitated with anti-DOK3 antibody and immunoblotted for Grb2 and DOK3. (C) Tolerant wild type and DOK3-deficient BMM were re-stimulated with 1 µg/ml LPS and lysates immunoblotted for SOS1 and JNK (loading control). Data are representative of three independent experiments.

    Techniques Used: Immunoprecipitation

    33) Product Images from "Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages"

    Article Title: Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080843

    Quercetin, but not Q3GA, inhibited LPS-induced JNK activation. (A and B) Effects of quercetin on the activation of MAP kinases (A) and NFκB pathway (B) in the RAW264 cells treated with LPS for 30 min. (C) Effects of quercetin and Q3GA on the activation of JNK pathway in the RAW264 cells treated with LPS for 30 min. (D) Identification of the signaling pathways responsible for the LPS-induced COX-2 expression in the RAW264 cells. Upper, effects of inhibitors for several signaling pathways on the LPS-induced COX-2 expression in the RAW264 cells. Concentrations of inhibitors are as follows: PD98059 (for MEK/ERK), 10 μM; SP600125 (for JNK), 10 μM; SB203580 (for p38), 10 μM; and wortmannin (for PI3K, 100 nM). Cells were treated with LPS (1 μg/ml) in the presence of inhibitors for 4 h. Lower , overexpression of wild-type JNK1 (WT) significantly induced the COX-2 expression, whereas a dominant negative mutant of JNK1 (DN) did not. (E) Upper, immunoblot analysis of JNK1 and JNK2 (β-actin as control) to confirm the knockdown. Lower, effects of knockdown of JNK1/2 on the inhibitory actions of quercetin (100 μM) on the LPS-induced COX-2 expression in the RAW264 cells. C, control siRNA; JNK1/2, a mixture of JNK1 and JNK2 siRNA. The ratios of COX-2 to β-actin were also presented. ND, not detected.
    Figure Legend Snippet: Quercetin, but not Q3GA, inhibited LPS-induced JNK activation. (A and B) Effects of quercetin on the activation of MAP kinases (A) and NFκB pathway (B) in the RAW264 cells treated with LPS for 30 min. (C) Effects of quercetin and Q3GA on the activation of JNK pathway in the RAW264 cells treated with LPS for 30 min. (D) Identification of the signaling pathways responsible for the LPS-induced COX-2 expression in the RAW264 cells. Upper, effects of inhibitors for several signaling pathways on the LPS-induced COX-2 expression in the RAW264 cells. Concentrations of inhibitors are as follows: PD98059 (for MEK/ERK), 10 μM; SP600125 (for JNK), 10 μM; SB203580 (for p38), 10 μM; and wortmannin (for PI3K, 100 nM). Cells were treated with LPS (1 μg/ml) in the presence of inhibitors for 4 h. Lower , overexpression of wild-type JNK1 (WT) significantly induced the COX-2 expression, whereas a dominant negative mutant of JNK1 (DN) did not. (E) Upper, immunoblot analysis of JNK1 and JNK2 (β-actin as control) to confirm the knockdown. Lower, effects of knockdown of JNK1/2 on the inhibitory actions of quercetin (100 μM) on the LPS-induced COX-2 expression in the RAW264 cells. C, control siRNA; JNK1/2, a mixture of JNK1 and JNK2 siRNA. The ratios of COX-2 to β-actin were also presented. ND, not detected.

    Techniques Used: Activation Assay, Expressing, Over Expression, Dominant Negative Mutation

    34) Product Images from "The Role of Thymosin Beta 4 on Odontogenic Differentiation in Human Dental Pulp Cells"

    Article Title: The Role of Thymosin Beta 4 on Odontogenic Differentiation in Human Dental Pulp Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061960

    Effects of Tβ4 activation on the expression of MAPK pathways and transcription factors in HDPCs. Cells were incubated without or with Tβ4 peptide (1 µg/ml) in OM. (A) Phosphorylation of p38, JNK, and ERK MAPK was determined by Western blot analysis for 180 min. (B) Expression of the transcription factors Runx2 and Osterix was examined by RT-PCR for 180 min. Tβ4 activation enhanced the phosphorylation of p38, JNK, and ERK, and expression of transcription factors such as Runx2 and osterix. Data are representative of three independent experiments.
    Figure Legend Snippet: Effects of Tβ4 activation on the expression of MAPK pathways and transcription factors in HDPCs. Cells were incubated without or with Tβ4 peptide (1 µg/ml) in OM. (A) Phosphorylation of p38, JNK, and ERK MAPK was determined by Western blot analysis for 180 min. (B) Expression of the transcription factors Runx2 and Osterix was examined by RT-PCR for 180 min. Tβ4 activation enhanced the phosphorylation of p38, JNK, and ERK, and expression of transcription factors such as Runx2 and osterix. Data are representative of three independent experiments.

    Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis"

    Article Title: Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002185

    Macrophage exosomes induce Tyr phosphorylation and activation of MAP Kinases. A. 3–5 µg/ml of exosomes was given to naive J774 macrophages for the indicated time-points. Membranes were blotted with 4G10 anti-phosphotyrosine antibody. NILX, LPSX and LEISHX exosomes induce differential Tyr phosphorylation in naive macrophages maximizing at 1 h. Phosphorylations are stronger with LPSX and LEISHX compared to NILX. Arrowheads point to phosphorylations occurring following exosome stimulation. B. Naive macrophages were stimulated with exosomes or 100 ng/ml of LPS for 1 h. ERK and P38 MAP Kinases are phosphorylated after 1 h stimulation with NILX, LPSX and LEISHX exosomes. JNK is also phosphorylated after NILX and LPSX stimulation but not as strongly after LEISHX stimulation. The plots on the right side of each blot show average densitometric quantifications of 3 separate experiments, normalized against total protein and non-treated samples. Error bars show standard deviation.
    Figure Legend Snippet: Macrophage exosomes induce Tyr phosphorylation and activation of MAP Kinases. A. 3–5 µg/ml of exosomes was given to naive J774 macrophages for the indicated time-points. Membranes were blotted with 4G10 anti-phosphotyrosine antibody. NILX, LPSX and LEISHX exosomes induce differential Tyr phosphorylation in naive macrophages maximizing at 1 h. Phosphorylations are stronger with LPSX and LEISHX compared to NILX. Arrowheads point to phosphorylations occurring following exosome stimulation. B. Naive macrophages were stimulated with exosomes or 100 ng/ml of LPS for 1 h. ERK and P38 MAP Kinases are phosphorylated after 1 h stimulation with NILX, LPSX and LEISHX exosomes. JNK is also phosphorylated after NILX and LPSX stimulation but not as strongly after LEISHX stimulation. The plots on the right side of each blot show average densitometric quantifications of 3 separate experiments, normalized against total protein and non-treated samples. Error bars show standard deviation.

    Techniques Used: Activation Assay, Standard Deviation

    36) Product Images from "Telomerase reverse transcriptase promotes the proliferation of human laryngeal carcinoma cells through activation of the activator protein 1"

    Article Title: Telomerase reverse transcriptase promotes the proliferation of human laryngeal carcinoma cells through activation of the activator protein 1

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1344

    Effect of TERT overexpression with modulation of the p38 and ERK signaling pathways on c-Fos and c-Jun expression and activation in the HEp-2 laryngeal carcinoma cells. (A) Western blot analysis of the effect of TERT overexpression (Ad-sh-TERT) and silencing (Ad-Sh-TERT) on the p38 signaling pathway and c-Fos and c-Jun expression in the presence and absence of the p38 inhibitor, SB202190. (B) Western blot analysis of the effect of TERT overexpression (Ad-sh-TERT) and silencing (Ad-Sh-TERT) on the ERK signaling pathway and c-Fos and c-Jun expression in the presence and absence of the ERK inhibitor, U0126.
    Figure Legend Snippet: Effect of TERT overexpression with modulation of the p38 and ERK signaling pathways on c-Fos and c-Jun expression and activation in the HEp-2 laryngeal carcinoma cells. (A) Western blot analysis of the effect of TERT overexpression (Ad-sh-TERT) and silencing (Ad-Sh-TERT) on the p38 signaling pathway and c-Fos and c-Jun expression in the presence and absence of the p38 inhibitor, SB202190. (B) Western blot analysis of the effect of TERT overexpression (Ad-sh-TERT) and silencing (Ad-Sh-TERT) on the ERK signaling pathway and c-Fos and c-Jun expression in the presence and absence of the ERK inhibitor, U0126.

    Techniques Used: Over Expression, Expressing, Activation Assay, Western Blot

    37) Product Images from "Regulation of c-Fos Gene Expression by NF-?B: A p65 Homodimer Binding Site in Mouse Embryonic Fibroblasts but Not Human HEK293 Cells"

    Article Title: Regulation of c-Fos Gene Expression by NF-?B: A p65 Homodimer Binding Site in Mouse Embryonic Fibroblasts but Not Human HEK293 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084062

    PMA-induced activation of ERK/Elk-1 and IKK/p65/NF-κB occurs through independent pathways. (A) WT, IKKα −/− , IKKβ −/− , and IKKγ −/− MEFs were stimulated with PMA for various periods. (B) WT MEFs were pretreated with either DMSO (0.1%) or U0126 (3 µM) for 30 min and then stimulated with PMA (10 nM) for the indicated periods. Total cell lysates were prepared and subjected to western blotting analysis; the antibodies used are indicated. (C) The localization of p65 (green) and nuclear staining (blue) were determined by using fluorescence microscopy. (D) HEK293 cells transfected with the NF-κB luciferase reporter and theβ-galactosidase plasmid were pretreated with DMSO (0.1%) or U0126 (3 µM) for 30 min and then stimulated with PMA (10 nM) for 6 h. The luciferase activity derived from NF-κB activation was normalized relative to the transfection efficiency based on β-galactosidase. Data in (A)–(C) are representative of 3 independent experiments. Data in (D) are the means ± SEM from 3 independent experiments.
    Figure Legend Snippet: PMA-induced activation of ERK/Elk-1 and IKK/p65/NF-κB occurs through independent pathways. (A) WT, IKKα −/− , IKKβ −/− , and IKKγ −/− MEFs were stimulated with PMA for various periods. (B) WT MEFs were pretreated with either DMSO (0.1%) or U0126 (3 µM) for 30 min and then stimulated with PMA (10 nM) for the indicated periods. Total cell lysates were prepared and subjected to western blotting analysis; the antibodies used are indicated. (C) The localization of p65 (green) and nuclear staining (blue) were determined by using fluorescence microscopy. (D) HEK293 cells transfected with the NF-κB luciferase reporter and theβ-galactosidase plasmid were pretreated with DMSO (0.1%) or U0126 (3 µM) for 30 min and then stimulated with PMA (10 nM) for 6 h. The luciferase activity derived from NF-κB activation was normalized relative to the transfection efficiency based on β-galactosidase. Data in (A)–(C) are representative of 3 independent experiments. Data in (D) are the means ± SEM from 3 independent experiments.

    Techniques Used: Activation Assay, Western Blot, Staining, Fluorescence, Microscopy, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Derivative Assay

    Schematic summary of the transcriptional regulation of mouse c-Fos gene expression. In addition to the recognized ERK-stimulated pathways required for c-Fos expression, the binding of the p65 homodimer to the κB site and the functioning of p65 in a coordinated manner with the transcription factors Elk-1, SRF, and CREB is prerequisite for mouse PMA-induced c-Fos gene expression.
    Figure Legend Snippet: Schematic summary of the transcriptional regulation of mouse c-Fos gene expression. In addition to the recognized ERK-stimulated pathways required for c-Fos expression, the binding of the p65 homodimer to the κB site and the functioning of p65 in a coordinated manner with the transcription factors Elk-1, SRF, and CREB is prerequisite for mouse PMA-induced c-Fos gene expression.

    Techniques Used: Expressing, Binding Assay

    38) Product Images from "Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate"

    Article Title: Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088089

    Effect of micropatterning on migration and proliferation of VSMCs. (A) VSMCs at passage 10 were cultured on the flat or micropatterned substrate for 18-free medium and were then treated with 5% serum for an additional 24 h. Proliferation rates were measured by MTT assay. (data are presented as the mean ± SEM, n = 3; P -value indicates a significant difference by student's t-test) (B) The flat and micropatterned substrates were coated with laminin or fibronectin. VSMCs were cultured on the coated substrate for 48 h and then treated with LPA (10 µmol/L) for various time periods (0–30 min). Soluble lysates were subjected to Western blotting for AKT, ERK and GAPDH. (C) VSMCs at passage 10 were cultured on the flat or micropatterned substrate. They were then scratched with a micropipette tip to form a cell-free (wounded) area and incubated in the absence or presence of 5% serum for 24 h. The migrated distance was determined as averaged distance of each cell from the boundary of wound. (data are presented as the mean ± SEM, each cell was evaluated respectively; asterisks indicate a significant difference by student's t-test, ** P
    Figure Legend Snippet: Effect of micropatterning on migration and proliferation of VSMCs. (A) VSMCs at passage 10 were cultured on the flat or micropatterned substrate for 18-free medium and were then treated with 5% serum for an additional 24 h. Proliferation rates were measured by MTT assay. (data are presented as the mean ± SEM, n = 3; P -value indicates a significant difference by student's t-test) (B) The flat and micropatterned substrates were coated with laminin or fibronectin. VSMCs were cultured on the coated substrate for 48 h and then treated with LPA (10 µmol/L) for various time periods (0–30 min). Soluble lysates were subjected to Western blotting for AKT, ERK and GAPDH. (C) VSMCs at passage 10 were cultured on the flat or micropatterned substrate. They were then scratched with a micropipette tip to form a cell-free (wounded) area and incubated in the absence or presence of 5% serum for 24 h. The migrated distance was determined as averaged distance of each cell from the boundary of wound. (data are presented as the mean ± SEM, each cell was evaluated respectively; asterisks indicate a significant difference by student's t-test, ** P

    Techniques Used: Migration, Cell Culture, MTT Assay, Western Blot, Incubation

    39) Product Images from "Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models"

    Article Title: Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2014.3

    Mechanisms of action associated with carfilzomib in CML. ( a , b ) Cells were exposed to IC 50 values of carfilzomib for 1 h, followed by growth in drug-free medium for 24 h before being harvested and whole cell lysates prepared. ( a ) Western blot analysis for activated (phosphorylated) ERK, Stat5 and PI3K signaling pathways following exposure to carfilzomib, total levels of ERK, Stat5 and PI3K were used as a loading control. Densitometry analysis is expressed as a percentage of untreated control, corrected to loading controls and the average results across cell lines and primary cells are given. Phosphorylated ERK was significantly decreased following carfilzomib treatment ( P
    Figure Legend Snippet: Mechanisms of action associated with carfilzomib in CML. ( a , b ) Cells were exposed to IC 50 values of carfilzomib for 1 h, followed by growth in drug-free medium for 24 h before being harvested and whole cell lysates prepared. ( a ) Western blot analysis for activated (phosphorylated) ERK, Stat5 and PI3K signaling pathways following exposure to carfilzomib, total levels of ERK, Stat5 and PI3K were used as a loading control. Densitometry analysis is expressed as a percentage of untreated control, corrected to loading controls and the average results across cell lines and primary cells are given. Phosphorylated ERK was significantly decreased following carfilzomib treatment ( P

    Techniques Used: Western Blot

    40) Product Images from "Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung AdenocarcinomasBim Mediates Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Activating EGFR Mutations and its Up-Regulation is Attenuated by Secondary Resistant Mutations, T790M and a Novel L747SGefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires Pro-Apoptotic BH3-only Protein Bim and Can Be Enhanced by BH3 MimeticsWhy Do Cancer Cells Become "Addicted" to Oncogenic Epidermal Growth Factor Receptor?"

    Article Title: Induction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung AdenocarcinomasBim Mediates Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Activating EGFR Mutations and its Up-Regulation is Attenuated by Secondary Resistant Mutations, T790M and a Novel L747SGefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires Pro-Apoptotic BH3-only Protein Bim and Can Be Enhanced by BH3 MimeticsWhy Do Cancer Cells Become "Addicted" to Oncogenic Epidermal Growth Factor Receptor?

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0040294

    The Status of BIM Is Influenced by EGFR Signaling (A) BIM phosphorylation and protein levels are influenced by the ERK signaling pathway. H3255 cells were treated with a MEK inhibitor (PD98059, 50 μM; U0126, 10 μM), a PI3K inhibitor (LY294002, 50 μM; wortmannin, 100 nM every 2 h), both, or 100 nM erlotinib for 8 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. (B) NIH 3T3 cells transfected with wild-type EGFR cDNAs were serum-starved overnight and then stimulated with 100 ng/ml human EGF. After the indicated times, cells were harvested. Lysates were analyzed by immunoblotting, using the indicated antibodies. t, total protein; p, phospho-protein.
    Figure Legend Snippet: The Status of BIM Is Influenced by EGFR Signaling (A) BIM phosphorylation and protein levels are influenced by the ERK signaling pathway. H3255 cells were treated with a MEK inhibitor (PD98059, 50 μM; U0126, 10 μM), a PI3K inhibitor (LY294002, 50 μM; wortmannin, 100 nM every 2 h), both, or 100 nM erlotinib for 8 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. (B) NIH 3T3 cells transfected with wild-type EGFR cDNAs were serum-starved overnight and then stimulated with 100 ng/ml human EGF. After the indicated times, cells were harvested. Lysates were analyzed by immunoblotting, using the indicated antibodies. t, total protein; p, phospho-protein.

    Techniques Used: Transfection

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    SDS Page:

    Article Title: Geraniin inhibits TNF-α-induced impairments of osteogenesis through NF-κB and p38 MAPK signalling pathways in bone marrow stem cells
    Article Snippet: .. Thirty microgram proteins were separated using 10%–12% SDS-PAGE (Beyotime Institute of Biotechnology, China), transferred onto polyvinylidene difluoride membranes, blocked with 5% skimmed milk in TBS–Tween (0.2% Tween-20) for 2 hour at 37°C and then incubated with primary antibodies overnight at 4°C including anti-NF-κB/p65 (1:2000, Cell Signaling Technology, USA), anti-IкB-α (1:2000, Cell Signaling Technology, USA), anti-p38 MAPK(1:2000, Cell Signaling Technology, USA) and β-actin (Beyotime Institute of Biotechnology, China). .. Membranes were incubated with the appropriate secondary antibodies (Beyotime Institute of Biotechnology, China) conjugated with an IRDye 800CW.

    Blocking Assay:

    Article Title: Tubeimoside I Protects Dopaminergic Neurons Against Inflammation-Mediated Damage in Lipopolysaccharide (LPS)-Evoked Model of Parkinson’s Disease in Rats
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    P38 MAPK and <t>ERK</t> are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and <t>JNK</t> were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P
    Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of for 1 on EGF-induced activation of <t>EGFR</t> 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), <t>p-ERK</t> (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
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    P38 MAPK and ERK are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and JNK were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P

    Journal: The Journal of Cell Biology

    Article Title: Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs

    doi: 10.1083/jcb.201712113

    Figure Lengend Snippet: P38 MAPK and ERK are involved in TLR8 activation in cultured neurons. (A and B) WT neurons at 16 DIV (A) and Tlr7 −/− neurons at 5 DIV (B) were stimulated with CL075/poly dT for different time periods as indicated. Phosphorylation levels and total protein levels of ERK, P38, and JNK were assessed by immunoblotting. Results are representative of five independent experiments, and the data of each independent experiment is the mean of experimental duplication. The data are presented as the mean + SEM (error bars). *, P

    Article Snippet: Chemicals and antibodies The antibodies and reagents used in this study were as follows: GFP (A6455; rabbit; Invitrogen; ); MAP2 (AB5622; rabbit; EMD Millipore; ); MAP2 (M4403; mouse; Sigma-Aldrich; ); SMI-312R (SMI-312R; mouse; Covance; ); HA (3F10; rat; Roche; ); phospho-P38 MAPK (9211; rabbit; Cell Signaling Technology); rabbit polyclonal P38 MAPK antibody (9212; rabbit; Cell Signaling Technology); phospho-ERK (4376; rabbit; Cell Signaling Technology); ERK (4695; rabbit; Cell Signaling Technology); phospho-JNK (9251; rabbit; Cell Signaling Technology); JNK (9252; rabbit; Cell Signaling Technology); phospho-TAK1 (9339; rabbit; Cell Signaling Technology); GAPDH (sc-25778; rabbit; Santa Cruz Biotechnology, Inc.; ); NeuN (MAB377; mouse; EMD Millipore; ); HRP-conjugated secondary antibodies (GE Healthcare); and Alexa Fluor 488– and Alexa Fluor 594–conjugated secondary antibodies (Invitrogen).

    Techniques: Activation Assay, Cell Culture

    Effect of different allosteric MEKi combined with BGB‐283 and their impact on the MEK/ERK pathway in Calu‐6 cells. The antiproliferative effect of combining BGB‐283 and (A) PD‐0325901, (B) pimasertib, (C) trametinib, or (D) RO5126766 was evaluated by EOHSA analysis. The considered dose combinations with synergy found by Pacifico’s approach at significance level 0.05 are highlighted. (E) Immunoblot for pMEK, MEK, pERK, ERK, and GAPDH in Calu‐6 cells after 1‐ and 24‐h treatment with DMSO (‐), selumetinib, PD‐0325901, pimasertib, trametinib, and RO5126766 at the indicated concentrations. (F) Dose–response curve of MEK phosphorylation was determined by HTRF assay after 1‐h treatment of serial dilutions of different MEKi in Calu‐6 cells.

    Journal: Molecular Oncology

    Article Title: RAF dimer inhibition enhances the antitumor activity of MEK inhibitors in K‐RAS mutant tumors

    doi: 10.1002/1878-0261.12698

    Figure Lengend Snippet: Effect of different allosteric MEKi combined with BGB‐283 and their impact on the MEK/ERK pathway in Calu‐6 cells. The antiproliferative effect of combining BGB‐283 and (A) PD‐0325901, (B) pimasertib, (C) trametinib, or (D) RO5126766 was evaluated by EOHSA analysis. The considered dose combinations with synergy found by Pacifico’s approach at significance level 0.05 are highlighted. (E) Immunoblot for pMEK, MEK, pERK, ERK, and GAPDH in Calu‐6 cells after 1‐ and 24‐h treatment with DMSO (‐), selumetinib, PD‐0325901, pimasertib, trametinib, and RO5126766 at the indicated concentrations. (F) Dose–response curve of MEK phosphorylation was determined by HTRF assay after 1‐h treatment of serial dilutions of different MEKi in Calu‐6 cells.

    Article Snippet: Antibodies used were obtained commercially from the following sources: anti‐B‐RAF (SC‐5248), Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐C‐RAF (610152), BD Biosciences (San Jose, CA, USA); antibodies to MEK (9122), MEK1 (2352), phospho‐MEK1/2 (Ser217/221) (9154), ERK (4695), phospho‐ERK1/2 (Thr202/Tyr204) (4370), GAPDH (2118s), and anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody, Cell Signaling Technology (Danvers, MA, USA); and anti‐mouse IgG HRP‐linked secondary antibody (A0168), Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: HTRF Assay

    Combination of BGB‐283 and selumetinib effectively inhibited selumetinib‐induced pMEK accumulation and led to sustained pERK reduction. (A) Dose–response curve of MEK phosphorylation in Calu‐6 cells treated with 1 µ m selumetinib combined with increasing concentrations of BGB‐283 or vemurafenib for 1, 6, and 24 h. (B) Immunoblotting of B‐RAF, C‐RAF, pMEK, MEK, pERK, and ERK in Calu‐6 cells incubated with 1 µ m selumetinib alone or combined with 1 µ m BGB‐283 or 1 µ m vemurafenib after 1‐ to 48‐h treatment. (C) Biochemical activity of compound C against WT B‐RAF, B‐RAF V600E , C‐RAF, and EGFR. (D) Comparison of synergistic effect of MEKi with BGB‐283 or compound C from P ‐value, the percentage of dose combination with synergy, and maximum EC 50 shift in Calu‐6 cells.

    Journal: Molecular Oncology

    Article Title: RAF dimer inhibition enhances the antitumor activity of MEK inhibitors in K‐RAS mutant tumors

    doi: 10.1002/1878-0261.12698

    Figure Lengend Snippet: Combination of BGB‐283 and selumetinib effectively inhibited selumetinib‐induced pMEK accumulation and led to sustained pERK reduction. (A) Dose–response curve of MEK phosphorylation in Calu‐6 cells treated with 1 µ m selumetinib combined with increasing concentrations of BGB‐283 or vemurafenib for 1, 6, and 24 h. (B) Immunoblotting of B‐RAF, C‐RAF, pMEK, MEK, pERK, and ERK in Calu‐6 cells incubated with 1 µ m selumetinib alone or combined with 1 µ m BGB‐283 or 1 µ m vemurafenib after 1‐ to 48‐h treatment. (C) Biochemical activity of compound C against WT B‐RAF, B‐RAF V600E , C‐RAF, and EGFR. (D) Comparison of synergistic effect of MEKi with BGB‐283 or compound C from P ‐value, the percentage of dose combination with synergy, and maximum EC 50 shift in Calu‐6 cells.

    Article Snippet: Antibodies used were obtained commercially from the following sources: anti‐B‐RAF (SC‐5248), Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐C‐RAF (610152), BD Biosciences (San Jose, CA, USA); antibodies to MEK (9122), MEK1 (2352), phospho‐MEK1/2 (Ser217/221) (9154), ERK (4695), phospho‐ERK1/2 (Thr202/Tyr204) (4370), GAPDH (2118s), and anti‐rabbit IgG horseradish peroxidase (HRP)‐linked secondary antibody, Cell Signaling Technology (Danvers, MA, USA); and anti‐mouse IgG HRP‐linked secondary antibody (A0168), Sigma‐Aldrich (St. Louis, MO, USA).

    Techniques: Incubation, Activity Assay

    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

    Schematic drawing showing (A) the experimental design with immediate (I), delayed (D) and no nerve repairs (N and DN) and (B) the sites where p-ERK 1/2 and cleaved caspase 3 were analyzed .

    Journal: BMC Neuroscience

    Article Title: Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats

    doi: 10.1186/1471-2202-12-12

    Figure Lengend Snippet: Schematic drawing showing (A) the experimental design with immediate (I), delayed (D) and no nerve repairs (N and DN) and (B) the sites where p-ERK 1/2 and cleaved caspase 3 were analyzed .

    Article Snippet: p-ERK 1/2 The sections were air dried, washed in PBS for 5 min, and thereafter incubated with a primary rabbit-anti-p-ERK1/2 antibody (9101, Cell Signaling technology, USA) at a dilution of 1:500 in 0.25% Triton-X-100 (Sigma-Aldrich,USA) and 0.25% bovine serum albumin (BSA; Sigma-Aldrich,USA) in PBS overnight at 4° C. The sections were then washed three times (5 min per wash) and incubated with the secondary Alexa flour 488 goat-anti-rabbit antibody (Molecular Probes, Eugene, Oregon, USA) at a dilution of 1:500 in 0.25% Triton-X-100 and 0.25% BSA in PBS for 1 hour at room temperature.

    Techniques:

    Graphs of regression analyses between length of neurofilaments (mm, dependent) and p-ERK 1/2 and cleaved caspase 3 (independent) at site of lesion and in distal nerve segment . For p-values and r 2  see Results.

    Journal: BMC Neuroscience

    Article Title: Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats

    doi: 10.1186/1471-2202-12-12

    Figure Lengend Snippet: Graphs of regression analyses between length of neurofilaments (mm, dependent) and p-ERK 1/2 and cleaved caspase 3 (independent) at site of lesion and in distal nerve segment . For p-values and r 2 see Results.

    Article Snippet: p-ERK 1/2 The sections were air dried, washed in PBS for 5 min, and thereafter incubated with a primary rabbit-anti-p-ERK1/2 antibody (9101, Cell Signaling technology, USA) at a dilution of 1:500 in 0.25% Triton-X-100 (Sigma-Aldrich,USA) and 0.25% bovine serum albumin (BSA; Sigma-Aldrich,USA) in PBS overnight at 4° C. The sections were then washed three times (5 min per wash) and incubated with the secondary Alexa flour 488 goat-anti-rabbit antibody (Molecular Probes, Eugene, Oregon, USA) at a dilution of 1:500 in 0.25% Triton-X-100 and 0.25% BSA in PBS for 1 hour at room temperature.

    Techniques: