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  • 99
    Name:
    ERK 1 Antibody
    Description:
    Anti ERK 1 Antibody G 8 is a mouse monoclonal IgG2b ERK 1 antibody provided at 200 µg ml specific for an epitope mapping between amino acids 291 335 within subdomain XI of ERK 1 of rat origin Anti ERK 1 Antibody G 8 is recommended for detection of ERK 1 p44 of mouse rat and human origin by WB IP IF IHC P and ELISA also reactive with additional species including and equine canine bovine and porcine Anti ERK 1 Antibody G 8 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems blocking peptide sc 271269 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of ERK 1 G 8 sc 271269
    Catalog Number:
    sc-271269
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Kinases and Phosphatases ERK 1 Antibodies ERK 1 Antibody G 8
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    Structured Review

    Santa Cruz Biotechnology erk
    TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against <t>ERK</t> as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against <t>Rictor</t> as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.
    Anti ERK 1 Antibody G 8 is a mouse monoclonal IgG2b ERK 1 antibody provided at 200 µg ml specific for an epitope mapping between amino acids 291 335 within subdomain XI of ERK 1 of rat origin Anti ERK 1 Antibody G 8 is recommended for detection of ERK 1 p44 of mouse rat and human origin by WB IP IF IHC P and ELISA also reactive with additional species including and equine canine bovine and porcine Anti ERK 1 Antibody G 8 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems blocking peptide sc 271269 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of ERK 1 G 8 sc 271269
    https://www.bioz.com/result/erk/product/Santa Cruz Biotechnology
    Average 99 stars, based on 136 article reviews
    Price from $9.99 to $1999.99
    erk - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Differential IKK/NF-κB Activity is Mediated by TSC2 through mTORC1 in PTEN-null Prostate Cancer and Tuberous Sclerosis Complex Tumor Cells"

    Article Title: Differential IKK/NF-κB Activity is Mediated by TSC2 through mTORC1 in PTEN-null Prostate Cancer and Tuberous Sclerosis Complex Tumor Cells

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-15-0213

    TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.
    Figure Legend Snippet: TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.

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

    TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.
    Figure Legend Snippet: TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.

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

    2) Product Images from "Ginsenoside Rg1 Drives Stimulations of Timosaponin AIII-Induced Anticancer Effects in Human Osteosarcoma Cells"

    Article Title: Ginsenoside Rg1 Drives Stimulations of Timosaponin AIII-Induced Anticancer Effects in Human Osteosarcoma Cells

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2020/8980124

    Effects of the combination of Rg1 and TA3 on signaling molecules in MG63 human osteosarcoma cells. (a) Activation of JNK, ERK, and p38, which are the representative MAPKs, is synergistically attenuated by Rg1 and TA3. (b) β -catenin and active form of CREB (p-CREB) were synergistically suppressed by Rg1 and TA3.
    Figure Legend Snippet: Effects of the combination of Rg1 and TA3 on signaling molecules in MG63 human osteosarcoma cells. (a) Activation of JNK, ERK, and p38, which are the representative MAPKs, is synergistically attenuated by Rg1 and TA3. (b) β -catenin and active form of CREB (p-CREB) were synergistically suppressed by Rg1 and TA3.

    Techniques Used: Activation Assay

    3) Product Images from "CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity"

    Article Title: CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20132349

    CARD9 mediates Curdlan-induced ERK activation but is not dispensable for NF-κB. (A and B) RAW264.7 cells stably expressing human Dectin-1, Dectin-2, or mock were stimulated with plate-coated curdlan (50 µg/ml) or α-mannans (40 µg/ml) for 1 h for preparing nuclear extract (A) or 30 min for preparing cell lysate (B), and then subjected to immunoblotting analysis using the indicated antibodies. (C–F) WT and CARD9-deficient BMDMs (C and E) or BMDCs (D and F) were stimulated with plate-coated curdlan (50 µg/ml) or α-mannans (40 µg/ml) for the indicated times for preparing cell lysate (C and D) and 1 h for preparing nuclear extract (E and F). The cell lysates or nuclear extracts were subjected to immunoblotting analysis using the indicated antibodies. Data shown are representative of three independent and reproducible experiments.
    Figure Legend Snippet: CARD9 mediates Curdlan-induced ERK activation but is not dispensable for NF-κB. (A and B) RAW264.7 cells stably expressing human Dectin-1, Dectin-2, or mock were stimulated with plate-coated curdlan (50 µg/ml) or α-mannans (40 µg/ml) for 1 h for preparing nuclear extract (A) or 30 min for preparing cell lysate (B), and then subjected to immunoblotting analysis using the indicated antibodies. (C–F) WT and CARD9-deficient BMDMs (C and E) or BMDCs (D and F) were stimulated with plate-coated curdlan (50 µg/ml) or α-mannans (40 µg/ml) for the indicated times for preparing cell lysate (C and D) and 1 h for preparing nuclear extract (E and F). The cell lysates or nuclear extracts were subjected to immunoblotting analysis using the indicated antibodies. Data shown are representative of three independent and reproducible experiments.

    Techniques Used: Activation Assay, Stable Transfection, Expressing

    4) Product Images from "Synthesis, Biological Evaluation and Mechanism Studies of Deoxytylophorinine and Its Derivatives as Potential Anticancer Agents"

    Article Title: Synthesis, Biological Evaluation and Mechanism Studies of Deoxytylophorinine and Its Derivatives as Potential Anticancer Agents

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030342

    Effects of 1, 9, 12, 16, 32, 33, and 35 on phosphorylated and total proteins of Akt and ERK in A549 cells. A549 cells were untreated or treated with 500 nM of 1, 9, 12, 16, 32, 33, and 35 for 24 h. Following 24 h of recovery, cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE. Immunoblots of cellular lysates were analyzed by antibodies of phosphorylated and total proteins of Akt and ERK, with the expression of β-actin as an internal control. A. one of the selected immunoblot analysis results of pAkt, Akt, pERK, ERK and β-actin. B. densitometric analysis results of pAkt, Akt, pERK, and ERK normalized to β-actin expression. Each histogram represents the mean values ± standard deviation of three dependent experiments. (*, p
    Figure Legend Snippet: Effects of 1, 9, 12, 16, 32, 33, and 35 on phosphorylated and total proteins of Akt and ERK in A549 cells. A549 cells were untreated or treated with 500 nM of 1, 9, 12, 16, 32, 33, and 35 for 24 h. Following 24 h of recovery, cell lysates were prepared and equal amounts of protein were analyzed by SDS-PAGE. Immunoblots of cellular lysates were analyzed by antibodies of phosphorylated and total proteins of Akt and ERK, with the expression of β-actin as an internal control. A. one of the selected immunoblot analysis results of pAkt, Akt, pERK, ERK and β-actin. B. densitometric analysis results of pAkt, Akt, pERK, and ERK normalized to β-actin expression. Each histogram represents the mean values ± standard deviation of three dependent experiments. (*, p

    Techniques Used: SDS Page, Western Blot, Expressing, Standard Deviation

    5) Product Images from "MLK4? functions as a negative regulator of MAPK signaling and cell invasion"

    Article Title: MLK4? functions as a negative regulator of MAPK signaling and cell invasion

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2012.6

    A schematic diagram illustrating the role of MLK4β in MAPK signaling. MLK3, upon activation, activates the p38, JNK and ERK MAPK pathways leading to different cellular responses, including invasion. Our results indicate that MLK4β negatively regulates MLK3 activation and the ERK, JNK and p38 MAPK-signaling pathways. Therefore, MLK4β-dependent suppression of MAPK signaling may be mediated through inhibition of MLK3 activation.
    Figure Legend Snippet: A schematic diagram illustrating the role of MLK4β in MAPK signaling. MLK3, upon activation, activates the p38, JNK and ERK MAPK pathways leading to different cellular responses, including invasion. Our results indicate that MLK4β negatively regulates MLK3 activation and the ERK, JNK and p38 MAPK-signaling pathways. Therefore, MLK4β-dependent suppression of MAPK signaling may be mediated through inhibition of MLK3 activation.

    Techniques Used: Activation Assay, Inhibition

    MLK4β negatively regulates p38, ERK and JNK signaling. ( a ) HCT116 cells were transfected with MLK3, MLK4 and nonspecific siRNA oligos. Cell lysates were immunoblotted with MLK4β, MLK3, p-p38, p38 and β-Actin antibodies. ( b ) Lysates from HCT116 cells transiently expressing pCMV5-FLAG, FLAG-MLK3 or FLAG-MLK4β were prepared and immunoblotted with FLAG, p-p38 and p38 antibodies. ( c ) HCT116 cells were transfected with nonspecific, MLK3 or MLK4 siRNA, and then treated with 0.5 M sorbitol for 30 min. Cell lysates were immunoblotted with MLK4β, MLK3, p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-Actin antibodies.
    Figure Legend Snippet: MLK4β negatively regulates p38, ERK and JNK signaling. ( a ) HCT116 cells were transfected with MLK3, MLK4 and nonspecific siRNA oligos. Cell lysates were immunoblotted with MLK4β, MLK3, p-p38, p38 and β-Actin antibodies. ( b ) Lysates from HCT116 cells transiently expressing pCMV5-FLAG, FLAG-MLK3 or FLAG-MLK4β were prepared and immunoblotted with FLAG, p-p38 and p38 antibodies. ( c ) HCT116 cells were transfected with nonspecific, MLK3 or MLK4 siRNA, and then treated with 0.5 M sorbitol for 30 min. Cell lysates were immunoblotted with MLK4β, MLK3, p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-Actin antibodies.

    Techniques Used: Transfection, Expressing

    6) Product Images from "Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation"

    Article Title: Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-61

    Prostate cell lines vary in oncogenic ETS expression and RAS/ERK pathway activation. (A) The sensitivity and specificity of antibodies detecting oncogenic ETS proteins were tested by immunoblot of the indicated amount of purified full-length proteins. (B) Immunoblots show levels of four oncogenic ETS proteins, pAKT (PI3K/AKT activation), pERK (RAS/ERK activation), total ERK, and tubulin control in six prostate cancer cell lines (left) and three cell lines derived from normal prostate (right). (C) Immunoblots show levels of ETV4 and pERK in the indicated cell lines with or without U0126 (10 μM, 10 hr). The same cell extracts are loaded on one gel above the dashed line, and a second gel below. ETV4 is only visible in DU145 cells after a very long exposure, hence it is not observed in (A) .
    Figure Legend Snippet: Prostate cell lines vary in oncogenic ETS expression and RAS/ERK pathway activation. (A) The sensitivity and specificity of antibodies detecting oncogenic ETS proteins were tested by immunoblot of the indicated amount of purified full-length proteins. (B) Immunoblots show levels of four oncogenic ETS proteins, pAKT (PI3K/AKT activation), pERK (RAS/ERK activation), total ERK, and tubulin control in six prostate cancer cell lines (left) and three cell lines derived from normal prostate (right). (C) Immunoblots show levels of ETV4 and pERK in the indicated cell lines with or without U0126 (10 μM, 10 hr). The same cell extracts are loaded on one gel above the dashed line, and a second gel below. ETV4 is only visible in DU145 cells after a very long exposure, hence it is not observed in (A) .

    Techniques Used: Expressing, Activation Assay, Purification, Western Blot, Derivative Assay

    7) Product Images from "Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling"

    Article Title: Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20041817

    Dok-1 and Dok-2 are irrelevant to TLR9, TLR3, or TLR2 signaling. (A–C) Activation of each MAP kinase was examined as in Fig. 2 A upon CpG ODN, poly(I:C), or Pam 3 CSK 4 treatment of BM-derived macrophages from mice. (D) Dok-1 or Dok-2 immunoprecipitates (IP) were subjected to immunoblotting (IB) for Dok-1, Dok-2, or phosphotyrosine (PY-Dok-1 or PY-Dok-2) upon CpG ODN, poly(I:C), or Pam 3 CSK 4 treatment of wild-type peritoneal macrophages for 30 min. Whole cell lysates (WCL) from these macrophages were subjected to immunoblotting for activated Erk (p-Erk) or total Erk as controls.
    Figure Legend Snippet: Dok-1 and Dok-2 are irrelevant to TLR9, TLR3, or TLR2 signaling. (A–C) Activation of each MAP kinase was examined as in Fig. 2 A upon CpG ODN, poly(I:C), or Pam 3 CSK 4 treatment of BM-derived macrophages from mice. (D) Dok-1 or Dok-2 immunoprecipitates (IP) were subjected to immunoblotting (IB) for Dok-1, Dok-2, or phosphotyrosine (PY-Dok-1 or PY-Dok-2) upon CpG ODN, poly(I:C), or Pam 3 CSK 4 treatment of wild-type peritoneal macrophages for 30 min. Whole cell lysates (WCL) from these macrophages were subjected to immunoblotting for activated Erk (p-Erk) or total Erk as controls.

    Techniques Used: Activation Assay, Derivative Assay, Mouse Assay

    Dok-1 and Dok-2 are negative regulators of Erk upon TLR4 signaling. (A) Total Erk, activated Erk (p-Erk), JNK (p-JNK), or p38 MAP kinase (p-p38) was examined with immunoblotting upon LPS treatment of BM-derived macrophages from mice. (B) NF-κB activation was assessed by immunoblotting for IκBα or its phosphorylation (p-IκBα) upon LPS treatment of macrophages in A. Control immunoblotting for Erk was performed. (C) NF-κB activity was examined by gel mobility shift assay upon LPS treatment of peritoneal macrophages. Positions of the NF-κB complex and the free probes are indicated. The specificity was determined by adding excess amounts of unlabeled competitor of the probe (LPS + comp) or not (LPS) to nuclear extracts of wild-type macrophages. (D) Activated Erk (p-Erk), total Erk, Dok-1, or Dok-2 was examined with immunoblotting upon LPS treatment of RAW 264.7 cells (mock) or those expressing exogenous Dok-1 (top) or Dok-2 (bottom). An arrowhead indicates the position of endogenous Dok-1 or Dok-2. (E) RAW 264.7 cells (mock) or those expressing exogenous Dok-1 or a Dok-1 mutant (Dok-1 YF) were examined as in D. (F) RAW 264.7 cells (mock) or those expressing exogenous Dok-1, Dok-1 YF, or Dok-2 were cultured in the presence (+) or absence (−) of LPS, and then the percentage of intracellular TNF-α + cells was determined with flow cytometry. SD is from triplicate experiments.
    Figure Legend Snippet: Dok-1 and Dok-2 are negative regulators of Erk upon TLR4 signaling. (A) Total Erk, activated Erk (p-Erk), JNK (p-JNK), or p38 MAP kinase (p-p38) was examined with immunoblotting upon LPS treatment of BM-derived macrophages from mice. (B) NF-κB activation was assessed by immunoblotting for IκBα or its phosphorylation (p-IκBα) upon LPS treatment of macrophages in A. Control immunoblotting for Erk was performed. (C) NF-κB activity was examined by gel mobility shift assay upon LPS treatment of peritoneal macrophages. Positions of the NF-κB complex and the free probes are indicated. The specificity was determined by adding excess amounts of unlabeled competitor of the probe (LPS + comp) or not (LPS) to nuclear extracts of wild-type macrophages. (D) Activated Erk (p-Erk), total Erk, Dok-1, or Dok-2 was examined with immunoblotting upon LPS treatment of RAW 264.7 cells (mock) or those expressing exogenous Dok-1 (top) or Dok-2 (bottom). An arrowhead indicates the position of endogenous Dok-1 or Dok-2. (E) RAW 264.7 cells (mock) or those expressing exogenous Dok-1 or a Dok-1 mutant (Dok-1 YF) were examined as in D. (F) RAW 264.7 cells (mock) or those expressing exogenous Dok-1, Dok-1 YF, or Dok-2 were cultured in the presence (+) or absence (−) of LPS, and then the percentage of intracellular TNF-α + cells was determined with flow cytometry. SD is from triplicate experiments.

    Techniques Used: Derivative Assay, Mouse Assay, Activation Assay, Activity Assay, Mobility Shift, Expressing, Mutagenesis, Cell Culture, Flow Cytometry, Cytometry

    8) Product Images from "Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells"

    Article Title: Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-647

    Lipid raft disruption inhibits the levels of pSrc, pFAK, pCav, pERK, pAkt, pPI3-K, and pAkt in breast carcinoma cell lines . (A) and (B) MDA-MB-231 and ZR 751 cells left untreated or pretreated with different concentrations of MβCD were subjected to Western blot analysis for total and phosphorylated forms of Src, FAK, Cav, ERK, PI3-K, and Akt. GAPDH was used to verify that similar amounts of protein were loaded in each lane. Densitometric analysis of phosphorylated molecules was done. The result provided is of a representative experiment repeated 3-4 times with concordant results (* p
    Figure Legend Snippet: Lipid raft disruption inhibits the levels of pSrc, pFAK, pCav, pERK, pAkt, pPI3-K, and pAkt in breast carcinoma cell lines . (A) and (B) MDA-MB-231 and ZR 751 cells left untreated or pretreated with different concentrations of MβCD were subjected to Western blot analysis for total and phosphorylated forms of Src, FAK, Cav, ERK, PI3-K, and Akt. GAPDH was used to verify that similar amounts of protein were loaded in each lane. Densitometric analysis of phosphorylated molecules was done. The result provided is of a representative experiment repeated 3-4 times with concordant results (* p

    Techniques Used: Multiple Displacement Amplification, Western Blot

    9) Product Images from "p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells"

    Article Title: p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011801

    Effects of knocking down adherens junction proteins on expression of other junctional proteins. DU145 cells were transfected with the indicated siRNAs for E-cadherin, P-cadherin, α-catenin and β-catenin, or with control siRNA (control), or transfection reagent only (mock). (A) After 72 h cells were lysed and protein levels of indicated proteins analysed by immunoblotting. Total ERK or GAPDH protein levels were used as a loading control. (B) Graphs show protein levels of P-cadherin, E-cadherin, α-catenin and p120ctn following knockdown of the indicated proteins , quantified by Odyssey scanning of immunoblots (left) or β-catenin knockdown, quantified by densitometry (right), from 4 independent experiments. Results are normalised to control. Bars represent SEM. *** p
    Figure Legend Snippet: Effects of knocking down adherens junction proteins on expression of other junctional proteins. DU145 cells were transfected with the indicated siRNAs for E-cadherin, P-cadherin, α-catenin and β-catenin, or with control siRNA (control), or transfection reagent only (mock). (A) After 72 h cells were lysed and protein levels of indicated proteins analysed by immunoblotting. Total ERK or GAPDH protein levels were used as a loading control. (B) Graphs show protein levels of P-cadherin, E-cadherin, α-catenin and p120ctn following knockdown of the indicated proteins , quantified by Odyssey scanning of immunoblots (left) or β-catenin knockdown, quantified by densitometry (right), from 4 independent experiments. Results are normalised to control. Bars represent SEM. *** p

    Techniques Used: Expressing, Transfection, Western Blot

    p120ctn depletion does not affect RhoA or Rac1 activity but increases Rap1 activity. DU145 cells were transfected with siRNAs for p120ctn (p120ctn (1), (2)), control siRNA (control) or with transfection reagent only (mock). After 72 h cells were lysed and incubated with GST-Rhotekin-RBD, GST-PAK1-PBD or GST-RalGDS-RBD on glutathione beads to pull down active RhoA, Rac1 and Rap1 respectively. Lysates of mock-transfected cells incubated with GTPγS to preload GTPases were used as a positive control for pulldown assays. (A) Example immunoblots for GTPase activity assays. ERK levels on immunoblots were used as a loading control. Ponceau staining of immunoblots shows the levels of GST-fusion proteins. (B) Graphs show data from 3 (RhoA, Rac1) or 4 (Rap1) independent experiments and results are compared to total RhoA, Rac1, Cdc42 and normalised to control. Error bars represent SEM. * p
    Figure Legend Snippet: p120ctn depletion does not affect RhoA or Rac1 activity but increases Rap1 activity. DU145 cells were transfected with siRNAs for p120ctn (p120ctn (1), (2)), control siRNA (control) or with transfection reagent only (mock). After 72 h cells were lysed and incubated with GST-Rhotekin-RBD, GST-PAK1-PBD or GST-RalGDS-RBD on glutathione beads to pull down active RhoA, Rac1 and Rap1 respectively. Lysates of mock-transfected cells incubated with GTPγS to preload GTPases were used as a positive control for pulldown assays. (A) Example immunoblots for GTPase activity assays. ERK levels on immunoblots were used as a loading control. Ponceau staining of immunoblots shows the levels of GST-fusion proteins. (B) Graphs show data from 3 (RhoA, Rac1) or 4 (Rap1) independent experiments and results are compared to total RhoA, Rac1, Cdc42 and normalised to control. Error bars represent SEM. * p

    Techniques Used: Activity Assay, Transfection, Incubation, Positive Control, Western Blot, Staining

    10) Product Images from "Sprouty2 and Spred1-2 Proteins Inhibit the Activation of the ERK Pathway Elicited by Cyclopentenone Prostanoids"

    Article Title: Sprouty2 and Spred1-2 Proteins Inhibit the Activation of the ERK Pathway Elicited by Cyclopentenone Prostanoids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016787

    Overexpression of hSprouty2 (hSpry2) inhibits cyP-elicited ERK/Elk-1 pathway activation. ( A ) HeLa cells transiently co-transfected with pCEFL-KZ-HA-ERK1 and either pCEFL-KZ-AU5-hSpry2 wt, pCEFL-KZ-AU5-hSpry2 Y55F, or pCEFL-KZ-AU5 (AU5-vector), were serum-starved for 18 h and then incubated with vehicle (-), 50 ng/ml FGF, or 10 µM PGA 1 (or 15d-PGJ 2 ), for 15 min. Cell lysates were immunoprecipitated with anti-HA mAb, and analyzed by immunoblot using anti-p-ERK and -HA antibodies. Results were similar in three additional experiments. The factor by which values of p-ERK increased is estimated as mean of four separate assays (in each case with a SD lower than 10% of mean). The expression levels of AU5-hSpry2 constructs were detected by immunoblotting whole cell lysates (WCL) with anti-AU5 mAb (lower panel). ( B ) HeLa cells were co-transfected with pcDNAIII-Gal4-Elk-1, pGal4-Luc, and pRL-TK together with pCEFL-KZ-AU5 containing the indicated hSpry2 constructs in (A), and also pCEFL-KZ-AU5-hSpry2 P59A P304A. The transfected cells were serum-starved for 18 h, incubated with vehicle (-), either 50 ng/ml FGF, or 10 µM PGA 1 , for 8 h, and then assayed for luciferase activity. The data are the mean and SD of three separate assays performed in triplicate (* vs AU5-vector + FGF, or + PGA 1 : p
    Figure Legend Snippet: Overexpression of hSprouty2 (hSpry2) inhibits cyP-elicited ERK/Elk-1 pathway activation. ( A ) HeLa cells transiently co-transfected with pCEFL-KZ-HA-ERK1 and either pCEFL-KZ-AU5-hSpry2 wt, pCEFL-KZ-AU5-hSpry2 Y55F, or pCEFL-KZ-AU5 (AU5-vector), were serum-starved for 18 h and then incubated with vehicle (-), 50 ng/ml FGF, or 10 µM PGA 1 (or 15d-PGJ 2 ), for 15 min. Cell lysates were immunoprecipitated with anti-HA mAb, and analyzed by immunoblot using anti-p-ERK and -HA antibodies. Results were similar in three additional experiments. The factor by which values of p-ERK increased is estimated as mean of four separate assays (in each case with a SD lower than 10% of mean). The expression levels of AU5-hSpry2 constructs were detected by immunoblotting whole cell lysates (WCL) with anti-AU5 mAb (lower panel). ( B ) HeLa cells were co-transfected with pcDNAIII-Gal4-Elk-1, pGal4-Luc, and pRL-TK together with pCEFL-KZ-AU5 containing the indicated hSpry2 constructs in (A), and also pCEFL-KZ-AU5-hSpry2 P59A P304A. The transfected cells were serum-starved for 18 h, incubated with vehicle (-), either 50 ng/ml FGF, or 10 µM PGA 1 , for 8 h, and then assayed for luciferase activity. The data are the mean and SD of three separate assays performed in triplicate (* vs AU5-vector + FGF, or + PGA 1 : p

    Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Expressing, Construct, Luciferase, Activity Assay

    hSpred1 and hSpred2 block PGA 1 -induced ERK/Elk-1 pathway activation. ( A ) HeLa cells transiently co-transfected with pCEFL-KZ-HA-ERK1 and either pCEFL-KZ-AU5-hSpry2 wt, pCEFL-KZ-AU5-hSpry2 P59A P304A, pCEFL-KZ-AU5-hSpred1, pCEFL-KZ-AU5-hSpred2, or pCEFL-KZ-AU5 (AU5-vector), were serum-starved for 18 h and then incubated with vehicle (-), either 100 ng/ml EGF, or 10 µM PGA 1 , for 15 min. Cell lysates were immunoprecipitated with anti-HA mAb and analyzed by immunoblot using anti-p-ERK and -HA antibodies. Results were similar in three additional experiments. The factor by which values of p-ERK increased is estimated as mean of four separate assays (in each case with a SD lower than 10% of mean). The expression levels of AU5-hSpry2, AU5-hSpred1, or AU5-hSpred2, constructs were detected by immunoblotting WCL with the corresponding mAb (lower panels). ( B ) HeLa cells were co-transfected with the plasmids pcDNAIII-Gal4-Elk-1, pGal4-Luc, and pRL-TK together with pCEFL-KZ-AU5 containing the indicated hSpry2, or hSpred1, or hSpred2, constructs denoted in (A). The transfected cells were serum-starved for 18 h, incubated with vehicle (-), either 100 ng/ml EGF, or 50 ng/ml FGF, or 10 µM PGA 1 , for 8 h, and then assayed for luciferase activity. The data are the mean and SD of three separate assays performed in triplicate (* vs AU5-vector + FGF, or AU5-vector + EGF, or + PGA 1 : p
    Figure Legend Snippet: hSpred1 and hSpred2 block PGA 1 -induced ERK/Elk-1 pathway activation. ( A ) HeLa cells transiently co-transfected with pCEFL-KZ-HA-ERK1 and either pCEFL-KZ-AU5-hSpry2 wt, pCEFL-KZ-AU5-hSpry2 P59A P304A, pCEFL-KZ-AU5-hSpred1, pCEFL-KZ-AU5-hSpred2, or pCEFL-KZ-AU5 (AU5-vector), were serum-starved for 18 h and then incubated with vehicle (-), either 100 ng/ml EGF, or 10 µM PGA 1 , for 15 min. Cell lysates were immunoprecipitated with anti-HA mAb and analyzed by immunoblot using anti-p-ERK and -HA antibodies. Results were similar in three additional experiments. The factor by which values of p-ERK increased is estimated as mean of four separate assays (in each case with a SD lower than 10% of mean). The expression levels of AU5-hSpry2, AU5-hSpred1, or AU5-hSpred2, constructs were detected by immunoblotting WCL with the corresponding mAb (lower panels). ( B ) HeLa cells were co-transfected with the plasmids pcDNAIII-Gal4-Elk-1, pGal4-Luc, and pRL-TK together with pCEFL-KZ-AU5 containing the indicated hSpry2, or hSpred1, or hSpred2, constructs denoted in (A). The transfected cells were serum-starved for 18 h, incubated with vehicle (-), either 100 ng/ml EGF, or 50 ng/ml FGF, or 10 µM PGA 1 , for 8 h, and then assayed for luciferase activity. The data are the mean and SD of three separate assays performed in triplicate (* vs AU5-vector + FGF, or AU5-vector + EGF, or + PGA 1 : p

    Techniques Used: Blocking Assay, Activation Assay, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Expressing, Construct, Luciferase, Activity Assay

    11) Product Images from "Activating transcription factor 2 (ATF2) controls tolfenamic acid-induced ATF3 expression via MAP kinase pathways"

    Article Title: Activating transcription factor 2 (ATF2) controls tolfenamic acid-induced ATF3 expression via MAP kinase pathways

    Journal: Oncogene

    doi: 10.1038/onc.2010.251

    Upstream kinase for ATF2 phosphorylation and ATF3 expression (A) Cells were treated with 30 µM of TA for the indicated times, and Western blot was performed for p-ERK, ERK, p-SAPK/JNK, SAPK/JNK, p-p38, and p38. (B) Cells were pre-treated with indicated concentrations of SB203585 and then treated with 30 µM of TA for 2 h. (C) Cells were pre-treated with U0126 (10 µM) or PD98059 (40 µM) and then treated with 30 µM of TA for 2 h. (D) Cells were pre-treated with SP600125 (30 µM) and then treated with 30 µM of TA for 2 h. (E) Cells were transfected with wild type (WT) and dominant negative (DN) mutant construct of ERK2 or p38 kinase and then treated with 30 µM of TA for 2 h. (F) Cells were transfected with pATF3-84/+34 and pRL-null vector and pre-treated with indicated concentrations (SB203580, 50 µM; U0126, 10 µM; SP600125, 30 µM) of inhibitors for 30 min and then treated with 30 µM of TA for 24 h. **, P
    Figure Legend Snippet: Upstream kinase for ATF2 phosphorylation and ATF3 expression (A) Cells were treated with 30 µM of TA for the indicated times, and Western blot was performed for p-ERK, ERK, p-SAPK/JNK, SAPK/JNK, p-p38, and p38. (B) Cells were pre-treated with indicated concentrations of SB203585 and then treated with 30 µM of TA for 2 h. (C) Cells were pre-treated with U0126 (10 µM) or PD98059 (40 µM) and then treated with 30 µM of TA for 2 h. (D) Cells were pre-treated with SP600125 (30 µM) and then treated with 30 µM of TA for 2 h. (E) Cells were transfected with wild type (WT) and dominant negative (DN) mutant construct of ERK2 or p38 kinase and then treated with 30 µM of TA for 2 h. (F) Cells were transfected with pATF3-84/+34 and pRL-null vector and pre-treated with indicated concentrations (SB203580, 50 µM; U0126, 10 µM; SP600125, 30 µM) of inhibitors for 30 min and then treated with 30 µM of TA for 24 h. **, P

    Techniques Used: Expressing, Western Blot, Transfection, Dominant Negative Mutation, Mutagenesis, Construct, Plasmid Preparation

    12) Product Images from "Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer"

    Article Title: Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109076

    Serum starvation suppresses GEM-induced PARP-1 degradation through inhibition of autophagy via the ERK signaling pathway. (A) KLM1 and KLM1-R cells were exposed to 10 μg/mL of GEM in the presence or absence of 20 μM of U0126 for the indicated time courses. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and LC3A/B. (B) and (C) KLM1 and KLM1-R cells were cultured in the medium with or without FBS and meanwhile exposed to either or both GEM and U0126 at the indicated concentration. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies. The arrow head indicates the mono-ADP ribosylated form of PARP-1. Arrows indicate the position area of cleaved caspase-3. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials.
    Figure Legend Snippet: Serum starvation suppresses GEM-induced PARP-1 degradation through inhibition of autophagy via the ERK signaling pathway. (A) KLM1 and KLM1-R cells were exposed to 10 μg/mL of GEM in the presence or absence of 20 μM of U0126 for the indicated time courses. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and LC3A/B. (B) and (C) KLM1 and KLM1-R cells were cultured in the medium with or without FBS and meanwhile exposed to either or both GEM and U0126 at the indicated concentration. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies. The arrow head indicates the mono-ADP ribosylated form of PARP-1. Arrows indicate the position area of cleaved caspase-3. The expression of PARP-1 was confirmed repeatedly by a distinct PARP-1 antibody described in Materials.

    Techniques Used: Inhibition, SDS Page, Cell Culture, Concentration Assay, Expressing

    Serum starvation induces activation and different localization of extracellular ERK between KLM1 and KLM1-R cells. (A) KLM1 and KLM1-R cells were cultured in medium with or without FBS or exposed to 10 μg/mL of GEM for 24 h. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and ERK. (B) and (C) The indicated cells were stained with specific antibodies against p-ERK, Hsp27 and LC3A/B after cells were cultured in medium with or without FBS for 24 h. DAPI: blue and p-ERK: red in (B) and LC3A/B: green and Hsp27: red in (C). Scale bar, 20 μm.
    Figure Legend Snippet: Serum starvation induces activation and different localization of extracellular ERK between KLM1 and KLM1-R cells. (A) KLM1 and KLM1-R cells were cultured in medium with or without FBS or exposed to 10 μg/mL of GEM for 24 h. Cell lysates were resolved by SDS-PAGE and probed with specific antibodies against p-ERK and ERK. (B) and (C) The indicated cells were stained with specific antibodies against p-ERK, Hsp27 and LC3A/B after cells were cultured in medium with or without FBS for 24 h. DAPI: blue and p-ERK: red in (B) and LC3A/B: green and Hsp27: red in (C). Scale bar, 20 μm.

    Techniques Used: Activation Assay, Cell Culture, SDS Page, Staining

    13) Product Images from "Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway"

    Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

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

    doi: 10.1016/j.neo.2018.10.009

    SDS22 inactivates AKT and MAPK–ERK signaling pathway through their dephosphorylation. (A) Whole cell lysates of MDA-MB-231 cells ectopically expressing either vector control or SDS22 for 48 hours were immunoblotted for the indicated proteins. (B) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells for the indicated proteins. Whole cell lysates were immunoprecipitated with either the IgG control or SDS22 antibody. (C) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells. Whole cell lysates were immunoprecipitated with either the IgG control or AKT/MEK/ERK antibody. Ratio of SDS22 and ERK/MEK/AKT was quantified by the Image J software. (D) Immunoblotting of whole cell lysates of MCF7 cells expressing either the vector control or wild-type SDS22 or mutants SDS22 for 48 hours. (E) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells ectopically expressing the indicated plasmids for 48 hours. Whole cell lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins.
    Figure Legend Snippet: SDS22 inactivates AKT and MAPK–ERK signaling pathway through their dephosphorylation. (A) Whole cell lysates of MDA-MB-231 cells ectopically expressing either vector control or SDS22 for 48 hours were immunoblotted for the indicated proteins. (B) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells for the indicated proteins. Whole cell lysates were immunoprecipitated with either the IgG control or SDS22 antibody. (C) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells. Whole cell lysates were immunoprecipitated with either the IgG control or AKT/MEK/ERK antibody. Ratio of SDS22 and ERK/MEK/AKT was quantified by the Image J software. (D) Immunoblotting of whole cell lysates of MCF7 cells expressing either the vector control or wild-type SDS22 or mutants SDS22 for 48 hours. (E) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells ectopically expressing the indicated plasmids for 48 hours. Whole cell lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins.

    Techniques Used: De-Phosphorylation Assay, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Immunoprecipitation, Software

    14) Product Images from "HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer"

    Article Title: HCRP-1 regulates EGFR–AKT–BIM-mediated anoikis resistance and serves as a prognostic marker in human colon cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1217-2

    Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P
    Figure Legend Snippet: Inhibition of HCRP-1 activates the EGFR/AKT signaling pathway and suppresses BIM protein expression. a , b HCT116 and SW620 cells were transfected with si-HCRP-1 or control siRNA, and then cells were harvested and submitted to Western blot detection for the protein expression of BIM, FoxO3a, EGFR, AKT, ERK, Mcl-1, Bcl-2, and β-actin. Phosphorylated forms of EGFR, AKT, FoxO3a, and ERK were also detected by western blot with the corresponding antibodies. c–f Cells were treatedwith CHX for 0, 0.5 , 1, and 2 h after transfection with si-HCRP-1 or control siRNA for 48 h, lysates obtained from these cells were submitted to western blot detection for the protein expression of EGFR. Error bars indicate mean ± SD. Every experiment was repeated at least three times. * P

    Techniques Used: Inhibition, Expressing, Transfection, Western Blot

    15) Product Images from "Elevated Serum Melatonin under Constant Darkness Enhances Neural Repair in Spinal Cord Injury through Regulation of Circadian Clock Proteins Expression"

    Article Title: Elevated Serum Melatonin under Constant Darkness Enhances Neural Repair in Spinal Cord Injury through Regulation of Circadian Clock Proteins Expression

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm8020135

    Constant dark condition accelerates excitatory synapse formation and axonal outgrowth in the rostral region via TrkB-ERK signaling pathway. The mid-thoracic segments (T6-8) were analyzed at POD 3. ( A ) The animals under LL condition showed hemorrhage in dorsal white column, interrupted neurite outgrowth, and massive loss of immature (DCX) and mature neurons (NeuN) after spinal cord injury. The DD condition promoted not only excitatory synaptic formation (PSD-95, nNOS, Nr2a ) but also axonal outgrowth (Tau) compared with the L/D group. VC, ventral commissure; Magnification = ×200; Scale bar = 40 μm; ( B ) TrkB-mediated ERK signal transduction was enhanced under DD condition, while LL condition decreased their activation in spite of the higher level of BDNF. Transcriptional regulation of Aanat gene was affected by the light/dark condition; ( C ) DD condition increased the core clock proteins (CKOCK, BMAL1, nuclear PER1) as well as mRNA ( Per1 ) in the rostral region. The level of melatonin receptor 1A (MT1) and 1B (MT2) was greater in the SCI + DD group than those of other groups. L/D, 12/12-h light/dark; LL, 24-h constant light; DD, 24-h constant dark. a p
    Figure Legend Snippet: Constant dark condition accelerates excitatory synapse formation and axonal outgrowth in the rostral region via TrkB-ERK signaling pathway. The mid-thoracic segments (T6-8) were analyzed at POD 3. ( A ) The animals under LL condition showed hemorrhage in dorsal white column, interrupted neurite outgrowth, and massive loss of immature (DCX) and mature neurons (NeuN) after spinal cord injury. The DD condition promoted not only excitatory synaptic formation (PSD-95, nNOS, Nr2a ) but also axonal outgrowth (Tau) compared with the L/D group. VC, ventral commissure; Magnification = ×200; Scale bar = 40 μm; ( B ) TrkB-mediated ERK signal transduction was enhanced under DD condition, while LL condition decreased their activation in spite of the higher level of BDNF. Transcriptional regulation of Aanat gene was affected by the light/dark condition; ( C ) DD condition increased the core clock proteins (CKOCK, BMAL1, nuclear PER1) as well as mRNA ( Per1 ) in the rostral region. The level of melatonin receptor 1A (MT1) and 1B (MT2) was greater in the SCI + DD group than those of other groups. L/D, 12/12-h light/dark; LL, 24-h constant light; DD, 24-h constant dark. a p

    Techniques Used: Transduction, Activation Assay

    Constant dark condition preserves excitatory circuits at the acute phase in the lumbar enlargement. The spinal segments caudal to the injury epicenter (L1-2) were analyzed at POD 3. ( A ) LL condition led to the reduction of the neuroglial markers (GFAP, NeuN) as well as extensive tissue damage in the secondary lesion. Both PSD-95 and Olig2 proteins were augmented when DD condition was applied. Neuronal degeneration found in histological examination could be an evidence supporting these results. VC, ventral commissure; Magnification = ×200; Scale bar = 40 μm; ( B ) Increased Aanat expression under DD condition activated TrkB-ERK signal transduction, but its repression under LL condition inhibited AKT-dependent intracellular signal; ( C ) LL condition decreased Per1 expression, nuclear distribution of PER1 protein, and glycosylated MT1 level, which was preserved under DD condition. L/D, 12/12-h light/dark; LL, 24-h constant light; DD, 24-h constant dark. a p
    Figure Legend Snippet: Constant dark condition preserves excitatory circuits at the acute phase in the lumbar enlargement. The spinal segments caudal to the injury epicenter (L1-2) were analyzed at POD 3. ( A ) LL condition led to the reduction of the neuroglial markers (GFAP, NeuN) as well as extensive tissue damage in the secondary lesion. Both PSD-95 and Olig2 proteins were augmented when DD condition was applied. Neuronal degeneration found in histological examination could be an evidence supporting these results. VC, ventral commissure; Magnification = ×200; Scale bar = 40 μm; ( B ) Increased Aanat expression under DD condition activated TrkB-ERK signal transduction, but its repression under LL condition inhibited AKT-dependent intracellular signal; ( C ) LL condition decreased Per1 expression, nuclear distribution of PER1 protein, and glycosylated MT1 level, which was preserved under DD condition. L/D, 12/12-h light/dark; LL, 24-h constant light; DD, 24-h constant dark. a p

    Techniques Used: Expressing, Transduction

    16) Product Images from "Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury: role of erythropoietin on progenitor cell mobilization"

    Article Title: Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury: role of erythropoietin on progenitor cell mobilization

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2008.160887

    Inhibition of EPO receptor (EPOR) in H9c2 myoblasts A , left, representative micrograph showing that EPOR was expressed in H9c2 cells. Right, negative picture shows the specificity of EPOR immunoreactivity demonstrated by preadsorption of EPOR antibody with blocking peptide. B , representative micrographs showing cell morphology by phase-contrast in control (untreated) cells, cells treated with 5 U ml −1 of EPO (+EPO), or combination with 5 U ml −1 of EPO and 30 ng ml −1 of EPOR antibody (+EPO/+EPOR-ab) at 400× magnification. Nuclei were counterstained with DAPI (blue). Horizontal bar = 10 μm. Bar graph shows cell viability in different treatments by LDH. C , representative blots in the upper panel showing pAkt, Akt (left), pERK, and ERK (right) expression. Lower bar graph shows the ratio of DU of phosphorylated and total protein for n = 4. * P
    Figure Legend Snippet: Inhibition of EPO receptor (EPOR) in H9c2 myoblasts A , left, representative micrograph showing that EPOR was expressed in H9c2 cells. Right, negative picture shows the specificity of EPOR immunoreactivity demonstrated by preadsorption of EPOR antibody with blocking peptide. B , representative micrographs showing cell morphology by phase-contrast in control (untreated) cells, cells treated with 5 U ml −1 of EPO (+EPO), or combination with 5 U ml −1 of EPO and 30 ng ml −1 of EPOR antibody (+EPO/+EPOR-ab) at 400× magnification. Nuclei were counterstained with DAPI (blue). Horizontal bar = 10 μm. Bar graph shows cell viability in different treatments by LDH. C , representative blots in the upper panel showing pAkt, Akt (left), pERK, and ERK (right) expression. Lower bar graph shows the ratio of DU of phosphorylated and total protein for n = 4. * P

    Techniques Used: Inhibition, Blocking Assay, Expressing

    17) Product Images from "MEKK1 Transduces Activin Signals in Keratinocytes To Induce Actin Stress Fiber Formation and Migration"

    Article Title: MEKK1 Transduces Activin Signals in Keratinocytes To Induce Actin Stress Fiber Formation and Migration

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.1.60-65.2005

    Activin B-induced, RhoA- and MEKK1-dependent JNK activity is essential, but p38 and protein syntheses are not required, for actin stress fiber formation. (A) Wild-type and Mekk1 ΔKD/ΔKD keratinocytes were treated with activin B for the indicated times, and cell lysates were analyzed for the phosphorylated isoforms and total JNK, p38, and ERK by Western blotting. (B) Wild-type keratinocytes were infected with retroviruses containing empty vector or RhoA(N19). The cells were treated with activin B for 10 min before being harvested for Western blot analyses, as described above. (C) Wild-type keratinocytes were pretreated with chemical inhibitors as indicated, followed by stimulation with activin B and TGFα for 1 h. Polymerized actins were detected by rhodamine-phalloidin staining, and nuclei were identified by DAPI staining. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; CHX, protein synthesis inhibitor CHX.
    Figure Legend Snippet: Activin B-induced, RhoA- and MEKK1-dependent JNK activity is essential, but p38 and protein syntheses are not required, for actin stress fiber formation. (A) Wild-type and Mekk1 ΔKD/ΔKD keratinocytes were treated with activin B for the indicated times, and cell lysates were analyzed for the phosphorylated isoforms and total JNK, p38, and ERK by Western blotting. (B) Wild-type keratinocytes were infected with retroviruses containing empty vector or RhoA(N19). The cells were treated with activin B for 10 min before being harvested for Western blot analyses, as described above. (C) Wild-type keratinocytes were pretreated with chemical inhibitors as indicated, followed by stimulation with activin B and TGFα for 1 h. Polymerized actins were detected by rhodamine-phalloidin staining, and nuclei were identified by DAPI staining. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; CHX, protein synthesis inhibitor CHX.

    Techniques Used: Activity Assay, Western Blot, Infection, Plasmid Preparation, Staining

    Both JNK and p38 are required for keratinocyte migration. (A) Primary mouse keratinocytes were used for an in vitro wound-healing assay. Cells were treated by activin B (10 ng/ml) or TGFα (5 ng/ml) in the presence of absence of various inhibitors at 5 μM. Pictures were taken at 0, 12, 24, and 36 h after wounding, and only the 24 h photos are shown. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; PD, ERK inhibitor PD98059; CHX, protein synthesis inhibitor CHX. (B) Model of molecular pathways by which activin signals induce actin stress fiber formation and keratinocyte migration. The activins cause activation of RhoA, which in turn induces the ROCK-dependent MEKK1-JNK pathway that leads to a transcription-independent actin stress fiber formation. ROCK, in regulating MLCK, might have a role independent of the MEKK1-JNK pathway in the control of actin cytoskeleton. The JNK might regulate actin stress fiber formation through phosphorylation of paxillin. Apart from actin stress fiber formation, this pathway also results in the induction of c-Jun phosphorylation, which might lead to transcription-dependent events. The activin signals transduce also through a RhoA-independent MEKK1-p38 pathway that is critical for the transcription-dependent migration. Neither pathway is connected to Smad activation.
    Figure Legend Snippet: Both JNK and p38 are required for keratinocyte migration. (A) Primary mouse keratinocytes were used for an in vitro wound-healing assay. Cells were treated by activin B (10 ng/ml) or TGFα (5 ng/ml) in the presence of absence of various inhibitors at 5 μM. Pictures were taken at 0, 12, 24, and 36 h after wounding, and only the 24 h photos are shown. SP, JNK inhibitor SP600125; SB, p38 inhibitor SB202190; PD, ERK inhibitor PD98059; CHX, protein synthesis inhibitor CHX. (B) Model of molecular pathways by which activin signals induce actin stress fiber formation and keratinocyte migration. The activins cause activation of RhoA, which in turn induces the ROCK-dependent MEKK1-JNK pathway that leads to a transcription-independent actin stress fiber formation. ROCK, in regulating MLCK, might have a role independent of the MEKK1-JNK pathway in the control of actin cytoskeleton. The JNK might regulate actin stress fiber formation through phosphorylation of paxillin. Apart from actin stress fiber formation, this pathway also results in the induction of c-Jun phosphorylation, which might lead to transcription-dependent events. The activin signals transduce also through a RhoA-independent MEKK1-p38 pathway that is critical for the transcription-dependent migration. Neither pathway is connected to Smad activation.

    Techniques Used: Migration, In Vitro, Wound Healing Assay, Activation Assay

    18) Product Images from "HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway"

    Article Title: HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2011.95

    Hypoxia requires PI3K/AKT, ERK, and STAT3 signaling to drive the maintenance of U251 cells. ( a ) PI3K/AKT, ERK/MAPK, and STAT3 signaling pathways were preferentially activated in U251SC. ( b ) U251 cells were treated with hypoxia and CoCl 2 (100
    Figure Legend Snippet: Hypoxia requires PI3K/AKT, ERK, and STAT3 signaling to drive the maintenance of U251 cells. ( a ) PI3K/AKT, ERK/MAPK, and STAT3 signaling pathways were preferentially activated in U251SC. ( b ) U251 cells were treated with hypoxia and CoCl 2 (100

    Techniques Used:

    19) Product Images from "The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release *"

    Article Title: The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.385542

    The ERK/NRF2 signaling pathway is associated with enhanced hCSC survival following preconditioning with CoPP. A , FACS analysis with annexin V/PI double staining shows that specific inhibition of ERK by U0126 diminishes the cytoprotective effect of CoPP
    Figure Legend Snippet: The ERK/NRF2 signaling pathway is associated with enhanced hCSC survival following preconditioning with CoPP. A , FACS analysis with annexin V/PI double staining shows that specific inhibition of ERK by U0126 diminishes the cytoprotective effect of CoPP

    Techniques Used: FACS, Double Staining, Inhibition

    20) Product Images from "Oridonin induces apoptosis and autophagy in murine fibrosarcoma L929 cells partly via NO-ERK-p53 positive-feedback loop signaling pathway"

    Article Title: Oridonin induces apoptosis and autophagy in murine fibrosarcoma L929 cells partly via NO-ERK-p53 positive-feedback loop signaling pathway

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2012.53

    Effects of ERK and p53 on oridonin-induced apoptosis and autophagy. Cells were incubated with 50 μmol/L oridonin for 24 h in the presence or absence of PD 98059 (PD) (5 μmol/L) or pifithrin-α (PFT-α) (5 μmol/L)
    Figure Legend Snippet: Effects of ERK and p53 on oridonin-induced apoptosis and autophagy. Cells were incubated with 50 μmol/L oridonin for 24 h in the presence or absence of PD 98059 (PD) (5 μmol/L) or pifithrin-α (PFT-α) (5 μmol/L)

    Techniques Used: Incubation

    Relationship between nitric oxide and ERK-p53. (A) Cells were incubated with 50 μmol/L oridonin for 24 h in the presence or absence of 1400w (10 μmol/L) or L-NAME (20 μmol/L) for 3 h and then analyzed by Western blotting to assess
    Figure Legend Snippet: Relationship between nitric oxide and ERK-p53. (A) Cells were incubated with 50 μmol/L oridonin for 24 h in the presence or absence of 1400w (10 μmol/L) or L-NAME (20 μmol/L) for 3 h and then analyzed by Western blotting to assess

    Techniques Used: Incubation, Western Blot

    21) Product Images from "Upregulation of RANTES Gene Expression in Neuroglia by Japanese Encephalitis Virus Infection"

    Article Title: Upregulation of RANTES Gene Expression in Neuroglia by Japanese Encephalitis Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.22.12107-12119.2004

    Induction of transcription factors depends on activation of ERK. After mock or JEV (NT113) infection, mixed glia were cultivated for a further 8 h in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The isolated nuclear extracts were subjected to the electrophoretic mobility shift assay with the A/B, E, NF-κB, and NF-IL-6 oligonucleotides. Similar results were obtained from three independent experiments.
    Figure Legend Snippet: Induction of transcription factors depends on activation of ERK. After mock or JEV (NT113) infection, mixed glia were cultivated for a further 8 h in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The isolated nuclear extracts were subjected to the electrophoretic mobility shift assay with the A/B, E, NF-κB, and NF-IL-6 oligonucleotides. Similar results were obtained from three independent experiments.

    Techniques Used: Activation Assay, Infection, Isolation, Electrophoretic Mobility Shift Assay

    Requirement of ERK activation for RANTES expression. After mock or JEV (NT113) infection, mixed glia were cultivated in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The level of RANTES in the supernatants (24 h postinfection) was determined by enzyme-linked immunosorbent assay ( n = 4) (A). The levels of phosphorylated ERK (8 h postinfection), total ERK (8 h postinfection), and JEV NS3 protein (24 h postinfection) were determined by Western blot analysis. One of three separate experiments is shown (B).
    Figure Legend Snippet: Requirement of ERK activation for RANTES expression. After mock or JEV (NT113) infection, mixed glia were cultivated in medium alone or in the presence of U0126 (10 μM) or NAC (5 mM). The level of RANTES in the supernatants (24 h postinfection) was determined by enzyme-linked immunosorbent assay ( n = 4) (A). The levels of phosphorylated ERK (8 h postinfection), total ERK (8 h postinfection), and JEV NS3 protein (24 h postinfection) were determined by Western blot analysis. One of three separate experiments is shown (B).

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

    22) Product Images from "Inhibition of 6‐phosphofructo‐2‐kinase suppresses fibroblast‐like synoviocytes‐mediated synovial inflammation and joint destruction in rheumatoid arthritis) Inhibition of 6‐phosphofructo‐2‐kinase suppresses fibroblast‐like synoviocytes‐mediated synovial inflammation and joint destruction in rheumatoid arthritis"

    Article Title: Inhibition of 6‐phosphofructo‐2‐kinase suppresses fibroblast‐like synoviocytes‐mediated synovial inflammation and joint destruction in rheumatoid arthritis) Inhibition of 6‐phosphofructo‐2‐kinase suppresses fibroblast‐like synoviocytes‐mediated synovial inflammation and joint destruction in rheumatoid arthritis

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13762

    Role of PFKFB3 in regulating activation of the NF‐κB and MAPK pathways. RA FLSs pretreated with the PFKFB3 inhibitor PFK15 (5 μM) for 4 h were stimulated with TNF‐α for 30 min. (A) Effect of PFK15 on the nuclear translocation of NF‐κB p65. Representative laser confocal microcopy images showing the effect of PFK15 on TNFα‐induced translocation of p65 (green stain) from three independent experiments. (B) Effect of PFK15 on IKK and IκBα phosphorylation. The lower panel shows a densitometric analysis of a Western blot from five independent experiments. (C) Effect of PFK15 on the phosphorylation of p38, JNK and ERK. The right panel shows a densitometric analysis of an immunoblot from five independent experiments. * P
    Figure Legend Snippet: Role of PFKFB3 in regulating activation of the NF‐κB and MAPK pathways. RA FLSs pretreated with the PFKFB3 inhibitor PFK15 (5 μM) for 4 h were stimulated with TNF‐α for 30 min. (A) Effect of PFK15 on the nuclear translocation of NF‐κB p65. Representative laser confocal microcopy images showing the effect of PFK15 on TNFα‐induced translocation of p65 (green stain) from three independent experiments. (B) Effect of PFK15 on IKK and IκBα phosphorylation. The lower panel shows a densitometric analysis of a Western blot from five independent experiments. (C) Effect of PFK15 on the phosphorylation of p38, JNK and ERK. The right panel shows a densitometric analysis of an immunoblot from five independent experiments. * P

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

    Involvement of lactate in the PFKFB3‐mediated activation of NF‐κB and MAPK pathways by RA FLSs. RA FLSs were treated with or without the PFKFB3 inhibitor PFK15 (5 μM) for 4 h and then incubated with or without lactic acid (Lac, 10 mM) for 6 h. The cells were stimulated with TNF‐α for 30 min before harvesting. (A) Effect of lactate treatment on nuclear translocation of NF‐κB p65. Representative laser confocal microcopy images showing the effect of lactate on TNFα‐induced translocation of p65 (green stain) from five independent experiments. (B) Effect of lactate on IKK and IκBα phosphorylation. The lower panel shows a densitometric analysis of Western blotting from five independent experiments. (C) Effect of lactate on the phosphorylation of p38, JNK and ERK. The right panel shows a densitometric analysis of immunoblot analyses from five independent experiments. * P
    Figure Legend Snippet: Involvement of lactate in the PFKFB3‐mediated activation of NF‐κB and MAPK pathways by RA FLSs. RA FLSs were treated with or without the PFKFB3 inhibitor PFK15 (5 μM) for 4 h and then incubated with or without lactic acid (Lac, 10 mM) for 6 h. The cells were stimulated with TNF‐α for 30 min before harvesting. (A) Effect of lactate treatment on nuclear translocation of NF‐κB p65. Representative laser confocal microcopy images showing the effect of lactate on TNFα‐induced translocation of p65 (green stain) from five independent experiments. (B) Effect of lactate on IKK and IκBα phosphorylation. The lower panel shows a densitometric analysis of Western blotting from five independent experiments. (C) Effect of lactate on the phosphorylation of p38, JNK and ERK. The right panel shows a densitometric analysis of immunoblot analyses from five independent experiments. * P

    Techniques Used: Activation Assay, Incubation, Translocation Assay, Staining, Western Blot

    23) Product Images from "Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis"

    Article Title: Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis

    Journal: Blood

    doi: 10.1182/blood-2016-09-739268

    Working model of p19 INK4d function during human terminal erythroid differentiation. The blue arrow denotes a “decreased” expression, whereas the red arrow denotes an “increased” expression. p19 INK4d knockdown increases PEBP1 expression and impairs the p -ERK-HSP70-GATA1 pathway, which delays human terminal erythroid differentiation and leads to generation of abnormal nucleus.
    Figure Legend Snippet: Working model of p19 INK4d function during human terminal erythroid differentiation. The blue arrow denotes a “decreased” expression, whereas the red arrow denotes an “increased” expression. p19 INK4d knockdown increases PEBP1 expression and impairs the p -ERK-HSP70-GATA1 pathway, which delays human terminal erythroid differentiation and leads to generation of abnormal nucleus.

    Techniques Used: Expressing

    p19 INK4d interacts with and negatively regulates PEBP1 and PEBP1 links p19 INK4d with the ERK pathway. (A) Representative images of silver-stained gels of IP proteins using IgG and p19 INK4d antibody. (Upper) Nonspecific bands; (lower) specific band is marked with a red arrow. (B) Representative electrospray ionization-MS and MS/MS profiling of a tryptic peptide that owns higher content. The top right corner is the amino acid sequence of this peptide and the specific amino acid sequence belongs to PEBP1 through identification of proteins from the protein database. (C) Representative immunofluorescence images showing p19 INK4d and PEBP1 localization in erythroblasts. DAPI was used to stain the nucleus. (D) Representative images of co-IP experiments with a p19 INK4d (left) or PEBP1 antibody (right). (E) Representative images of western blotting showing PEBP1 expression in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of western blotting showing PEBP1, p -ERK, ERK, HSP70, and GATA1 levels in erythroblasts transfected with PEBP1 siRNA or scramble siRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P
    Figure Legend Snippet: p19 INK4d interacts with and negatively regulates PEBP1 and PEBP1 links p19 INK4d with the ERK pathway. (A) Representative images of silver-stained gels of IP proteins using IgG and p19 INK4d antibody. (Upper) Nonspecific bands; (lower) specific band is marked with a red arrow. (B) Representative electrospray ionization-MS and MS/MS profiling of a tryptic peptide that owns higher content. The top right corner is the amino acid sequence of this peptide and the specific amino acid sequence belongs to PEBP1 through identification of proteins from the protein database. (C) Representative immunofluorescence images showing p19 INK4d and PEBP1 localization in erythroblasts. DAPI was used to stain the nucleus. (D) Representative images of co-IP experiments with a p19 INK4d (left) or PEBP1 antibody (right). (E) Representative images of western blotting showing PEBP1 expression in erythroblasts infected with Lucif shRNA or p19 INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of western blotting showing PEBP1, p -ERK, ERK, HSP70, and GATA1 levels in erythroblasts transfected with PEBP1 siRNA or scramble siRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P

    Techniques Used: Staining, Mass Spectrometry, Sequencing, Immunofluorescence, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, shRNA, Transfection

    24) Product Images from "Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway"

    Article Title: Protein Phosphatase 1 Regulatory Subunit SDS22 Inhibits Breast Cancer Cell Tumorigenesis by Functioning as a Negative Regulator of the AKT Signaling Pathway

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

    doi: 10.1016/j.neo.2018.10.009

    SDS22 inactivates AKT and MAPK–ERK signaling pathway through their dephosphorylation. (A) Whole cell lysates of MDA-MB-231 cells ectopically expressing either vector control or SDS22 for 48 hours were immunoblotted for the indicated proteins. (B) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells for the indicated proteins. Whole cell lysates were immunoprecipitated with either the IgG control or SDS22 antibody. (C) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells. Whole cell lysates were immunoprecipitated with either the IgG control or AKT/MEK/ERK antibody. Ratio of SDS22 and ERK/MEK/AKT was quantified by the Image J software. (D) Immunoblotting of whole cell lysates of MCF7 cells expressing either the vector control or wild-type SDS22 or mutants SDS22 for 48 hours. (E) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells ectopically expressing the indicated plasmids for 48 hours. Whole cell lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins.
    Figure Legend Snippet: SDS22 inactivates AKT and MAPK–ERK signaling pathway through their dephosphorylation. (A) Whole cell lysates of MDA-MB-231 cells ectopically expressing either vector control or SDS22 for 48 hours were immunoblotted for the indicated proteins. (B) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells for the indicated proteins. Whole cell lysates were immunoprecipitated with either the IgG control or SDS22 antibody. (C) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells. Whole cell lysates were immunoprecipitated with either the IgG control or AKT/MEK/ERK antibody. Ratio of SDS22 and ERK/MEK/AKT was quantified by the Image J software. (D) Immunoblotting of whole cell lysates of MCF7 cells expressing either the vector control or wild-type SDS22 or mutants SDS22 for 48 hours. (E) Immunoblotting of immunoprecipitates and input whole cell lysates of MCF7 cells ectopically expressing the indicated plasmids for 48 hours. Whole cell lysates were immunoprecipitated with FLAG antibody. The immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins.

    Techniques Used: De-Phosphorylation Assay, Multiple Displacement Amplification, Expressing, Plasmid Preparation, Immunoprecipitation, Software

    25) Product Images from "Visfatin Promotes IL-6 and TNF-α Production in Human Synovial Fibroblasts by Repressing miR-199a-5p through ERK, p38 and JNK Signaling Pathways"

    Article Title: Visfatin Promotes IL-6 and TNF-α Production in Human Synovial Fibroblasts by Repressing miR-199a-5p through ERK, p38 and JNK Signaling Pathways

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19010190

    Schema of signaling pathways involved in visfatin-induced increases in IL-6 and TNF-α expression in synovial fibroblasts. Visfatin promotes IL-6 and TNF-α production (red arrows) in human synovial fibroblasts by inhibiting miR-199a-5p expression (T bars) via the ERK, p38 and JNK signaling pathways (black arrows).
    Figure Legend Snippet: Schema of signaling pathways involved in visfatin-induced increases in IL-6 and TNF-α expression in synovial fibroblasts. Visfatin promotes IL-6 and TNF-α production (red arrows) in human synovial fibroblasts by inhibiting miR-199a-5p expression (T bars) via the ERK, p38 and JNK signaling pathways (black arrows).

    Techniques Used: Expressing

    Visfatin increases IL-6 and TNF-α expression via inhibition of miR-199a-5p through the ERK, p38 and JNK signaling pathways. ( A ) Searches of three online computational algorithms (TargetScan, miRWalk and miRanda) for candidate miRNAs that target the IL-6 and TNF-α regions revealed the involvement of miR-199a-5p; ( B ) OASFs were incubated with visfatin for 24 h; miR-199a-5p expression was assessed by qPCR; ( C – E ) OASFs were transfected with miR-199a-5p mimic for 24 h, followed by stimulation with visfatin for 24 h; IL-6 and TNF-α expression was examined by qPCR, Western blot and ELISA assay; ( F ) Schematic 3′-UTR representation of human IL-6 and TNF-α containing the miR-199a-5p binding site; ( G ) OASFs were transfected with indicated luciferase plasmids before incubation with visfatin for 24 h; Luciferase activity was assessed; ( H ) OASFs were pretreated with ERK, p38, and JNK inhibitors for 30 min followed by stimulation with visfatin (30 ng/mL) for 24 h; miR-199a-5p expression was examined by qPCR. Results are expressed as the mean ± SEM. * p
    Figure Legend Snippet: Visfatin increases IL-6 and TNF-α expression via inhibition of miR-199a-5p through the ERK, p38 and JNK signaling pathways. ( A ) Searches of three online computational algorithms (TargetScan, miRWalk and miRanda) for candidate miRNAs that target the IL-6 and TNF-α regions revealed the involvement of miR-199a-5p; ( B ) OASFs were incubated with visfatin for 24 h; miR-199a-5p expression was assessed by qPCR; ( C – E ) OASFs were transfected with miR-199a-5p mimic for 24 h, followed by stimulation with visfatin for 24 h; IL-6 and TNF-α expression was examined by qPCR, Western blot and ELISA assay; ( F ) Schematic 3′-UTR representation of human IL-6 and TNF-α containing the miR-199a-5p binding site; ( G ) OASFs were transfected with indicated luciferase plasmids before incubation with visfatin for 24 h; Luciferase activity was assessed; ( H ) OASFs were pretreated with ERK, p38, and JNK inhibitors for 30 min followed by stimulation with visfatin (30 ng/mL) for 24 h; miR-199a-5p expression was examined by qPCR. Results are expressed as the mean ± SEM. * p

    Techniques Used: Expressing, Inhibition, Incubation, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Activity Assay

    26) Product Images from "Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway"

    Article Title: Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway

    Journal: Scientific Reports

    doi: 10.1038/srep27080

    Phosphorylation of ERK by ROS is required of eIF2α phosphorylation and autophagy induction. ( a ) Caco-2 cells were incubated in the presence of rVvhA (50 pg/mL) for various times (0–6 h) and then harvested. Total protein was extracted and blotted with p-ERK, p-JNK, p-p38, ERK, JNK, and p38 antibodies. Data represent mean ± S.E. n = 4. * P
    Figure Legend Snippet: Phosphorylation of ERK by ROS is required of eIF2α phosphorylation and autophagy induction. ( a ) Caco-2 cells were incubated in the presence of rVvhA (50 pg/mL) for various times (0–6 h) and then harvested. Total protein was extracted and blotted with p-ERK, p-JNK, p-p38, ERK, JNK, and p38 antibodies. Data represent mean ± S.E. n = 4. * P

    Techniques Used: Incubation

    VvhA mediates intestinal inflammation and autophagy induced by V. vulinficus. ICR mice were inoculated intragastrically with WT, boiled WT (Cont), vvhA mutant, and vvhA complement (comp) at 1.1 × 10 9 CFU/mL for 18 h. n = 7. ( a ) Representative images of H E stained ileum tissues are shown. Damaged intestinal tissue is shown at higher magnification. Scale bar represents 100 μm. ( b ) The expression levels of autophagy markers in ileum tissue were determined by western blot. ( c ) Mice received intragastric inoculation of Cont, WT, vvhA mutant, and vvhA comp at 1.1 × 10 9 CFU/mL, and were sacrificed 18 h later. The FITC-dextran was orally administered into mice for 4 h before euthanasia. Analysis of FITC-dextran translocation into the blood stream by luminometry reveals intestinal paracellular permeability. n = 7. ( d ) A proposed model for VvhA-evoked signaling pathway in intestinal epithelial cells. VvhA plays an essential role in the dissemination and pathogenesis of V. vulnificus in intestinal cells, where VvhA induces autophagy-related cell death through lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2a-dependent autophagy activation.
    Figure Legend Snippet: VvhA mediates intestinal inflammation and autophagy induced by V. vulinficus. ICR mice were inoculated intragastrically with WT, boiled WT (Cont), vvhA mutant, and vvhA complement (comp) at 1.1 × 10 9 CFU/mL for 18 h. n = 7. ( a ) Representative images of H E stained ileum tissues are shown. Damaged intestinal tissue is shown at higher magnification. Scale bar represents 100 μm. ( b ) The expression levels of autophagy markers in ileum tissue were determined by western blot. ( c ) Mice received intragastric inoculation of Cont, WT, vvhA mutant, and vvhA comp at 1.1 × 10 9 CFU/mL, and were sacrificed 18 h later. The FITC-dextran was orally administered into mice for 4 h before euthanasia. Analysis of FITC-dextran translocation into the blood stream by luminometry reveals intestinal paracellular permeability. n = 7. ( d ) A proposed model for VvhA-evoked signaling pathway in intestinal epithelial cells. VvhA plays an essential role in the dissemination and pathogenesis of V. vulnificus in intestinal cells, where VvhA induces autophagy-related cell death through lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2a-dependent autophagy activation.

    Techniques Used: Mouse Assay, Mutagenesis, Staining, Expressing, Western Blot, Translocation Assay, Permeability, Activation Assay

    27) Product Images from "In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model"

    Article Title: In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model

    Journal: Scientific Reports

    doi: 10.1038/srep30418

    Effect of EV-MSC and EV-MSC/Rluc on LLC-1 effluc activity, and apoptosis mechanism. ( A , C ) Representative BLI of EV-MSC and MSC/Rluc-EV-treated LLC-effluc activity. The viability of LLC-effluc cells decreased with increasing concentration of EV-MSC and EV-MSC/Rluc. ( B , D ) Quantitative effluc activity of LLC-effluc cells. ( E ) Annexin V and PI staining for analyzing LLC cell apoptosis. The percentage of apoptotic cells was increased after treatment with 10 or 20 μg/mL of EV-MSC/Rluc for 24 h. ( F ) Representative western blot analyzing pERK1/2, cleavage of apoptosis markers caspase 3 and PARP levels in EV MSC/Rluc-treated LLC cells. The fold changes were normalized for pERK with total ERK. Cleaved PARP and cleaved caspase 3 normalized with β-actin. Data are expressed as the mean ± standard deviation (SD) of three independent experiments, *p
    Figure Legend Snippet: Effect of EV-MSC and EV-MSC/Rluc on LLC-1 effluc activity, and apoptosis mechanism. ( A , C ) Representative BLI of EV-MSC and MSC/Rluc-EV-treated LLC-effluc activity. The viability of LLC-effluc cells decreased with increasing concentration of EV-MSC and EV-MSC/Rluc. ( B , D ) Quantitative effluc activity of LLC-effluc cells. ( E ) Annexin V and PI staining for analyzing LLC cell apoptosis. The percentage of apoptotic cells was increased after treatment with 10 or 20 μg/mL of EV-MSC/Rluc for 24 h. ( F ) Representative western blot analyzing pERK1/2, cleavage of apoptosis markers caspase 3 and PARP levels in EV MSC/Rluc-treated LLC cells. The fold changes were normalized for pERK with total ERK. Cleaved PARP and cleaved caspase 3 normalized with β-actin. Data are expressed as the mean ± standard deviation (SD) of three independent experiments, *p

    Techniques Used: Activity Assay, Concentration Assay, Staining, Western Blot, Standard Deviation

    28) Product Images from "E3 Ubiquitin Ligase Cbl-b Prevents Tumor Metastasis by Maintaining the Epithelial Phenotype in Multiple Drug-Resistant Gastric and Breast Cancer Cells"

    Article Title: E3 Ubiquitin Ligase Cbl-b Prevents Tumor Metastasis by Maintaining the Epithelial Phenotype in Multiple Drug-Resistant Gastric and Breast Cancer Cells

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

    doi: 10.1016/j.neo.2017.01.011

    Overexpression of Cbl-b repressed the mesenchymal phenotype by the inhibition of ERK/Akt-miR-200c-ZEB1 axis. (A) SGC7901/Adr and MCF-7/Adr cells were transfected with Cbl-b WT and empty vector plasmid for 48 hours. The relative level of miR-200c was analyzed by qRT-PCR. * P
    Figure Legend Snippet: Overexpression of Cbl-b repressed the mesenchymal phenotype by the inhibition of ERK/Akt-miR-200c-ZEB1 axis. (A) SGC7901/Adr and MCF-7/Adr cells were transfected with Cbl-b WT and empty vector plasmid for 48 hours. The relative level of miR-200c was analyzed by qRT-PCR. * P

    Techniques Used: Over Expression, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR

    Overexpression of Cbl-b promoted the ubiquitination and degradation of EGFR and EGFR pathway inactivation. SGC7901/Adr and MCF-7/Adr cells were transfected with Cbl-b WT and empty vector plasmid for 48 hours. (A) EGFR and downstream ERK/Akt were analyzed by Western blot. (B) The interaction of Cbl-b and EGFR was detected by immunosedimentation and Western blot. (C) The ubiquitination of EGFR was detected by immunosedimentation and Western blot.
    Figure Legend Snippet: Overexpression of Cbl-b promoted the ubiquitination and degradation of EGFR and EGFR pathway inactivation. SGC7901/Adr and MCF-7/Adr cells were transfected with Cbl-b WT and empty vector plasmid for 48 hours. (A) EGFR and downstream ERK/Akt were analyzed by Western blot. (B) The interaction of Cbl-b and EGFR was detected by immunosedimentation and Western blot. (C) The ubiquitination of EGFR was detected by immunosedimentation and Western blot.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Western Blot

    Schematic representation of the proposed model. (A) The E3 ubiquitin ligase Cbl-b is expressed at low levels in high-invasive MDR gastric cancer and breast cancer cells. EGFR-activated ERK/Akt enhanced the expression of the E-cadherin transcription repressor ZEB1 through the downregulation of miR-200c. Downregulation of E-cadherin led to EMT and tumor metastasis. (B) Overexpression of Cbl-b inhibited EGFR and the downstream ERK/Akt signal by the ubiquitination and degradation of EGFR. Inactivation of the EGFR pathway decreased the expression of the E-cadherin transcription repressor ZEB1 through the upregulation of miR-200c. E-cadherin is upregulated, and EMT and tumor metastasis are repressed.
    Figure Legend Snippet: Schematic representation of the proposed model. (A) The E3 ubiquitin ligase Cbl-b is expressed at low levels in high-invasive MDR gastric cancer and breast cancer cells. EGFR-activated ERK/Akt enhanced the expression of the E-cadherin transcription repressor ZEB1 through the downregulation of miR-200c. Downregulation of E-cadherin led to EMT and tumor metastasis. (B) Overexpression of Cbl-b inhibited EGFR and the downstream ERK/Akt signal by the ubiquitination and degradation of EGFR. Inactivation of the EGFR pathway decreased the expression of the E-cadherin transcription repressor ZEB1 through the upregulation of miR-200c. E-cadherin is upregulated, and EMT and tumor metastasis are repressed.

    Techniques Used: Expressing, Over Expression

    29) Product Images from "Hepatitis B Virus e Antigen Activates the Suppressor of Cytokine Signaling 2 to Repress Interferon Action"

    Article Title: Hepatitis B Virus e Antigen Activates the Suppressor of Cytokine Signaling 2 to Repress Interferon Action

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01773-6

    The effect of HBeAg on regulation of SOCS2 expression and ERK signaling. ( A ) HepG2 cells were transfected with pCMV-HBeAg at different concentrations for 24 h. SOCS1, SOCS2, and SOCS3 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( B – E ) HepG2 cells were incubated with rHBeAg at different concentrations ( B ) or transfected with pCMV-HBeAg for different times ( C ). HepG2 cells were transfected with pCMV-Tag2B, pCMV-HBeAg, or pCMV-HBeAg-1896mut for 24 h ( D ) or incubated with PBS, rHBeAg, heat-inactivated HBeAg, or rHBcAg for 12 h ( E ). SOCS2 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( F ) HepG2, pBlue-SK-transfected HepG2, pHBV1.3-transfected HepG2, or HepG2.2.15 cells were harvested 24 h after transfection. SOCS2 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( G ) HepG2 cells were transfected with pBlue-SK or pHBV1.3 for different times. SOCS2 mRNA was measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( H ) HepG2 cells were transfected with pCMV-tag2B or plasmids expressing individual HBV proteins for 48 h. SOCS2 mRNA was determined by real-time PCR (upper). SOCS2 and β-actin mRNAs were determined by semi-quantitative RT-PCR (lower). ( I ) HepG2 cells were transfected with pBlue-SK, pHBV1.3, or pHBV1.3-1896mut for 24 h. SOCS2 mRNA was determined by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( J ) HepG2 cells were pretreated with each of specific kinase inhibitors for 12 h, and incubated with rHBeAg or PBS for 24 h. SOCS2 mRNAs were measured by real-time PCR. ( K ) HepG2 cells were incubated with PBS, rHBeAg, or 12-O-tetradecanoylphorbol 13-acetate (TPA) for 12 h. p-ERK, ERK, and GAPDH proteins were detected by Western blot analyses. Data shown are means ± SE; n = 3. *p
    Figure Legend Snippet: The effect of HBeAg on regulation of SOCS2 expression and ERK signaling. ( A ) HepG2 cells were transfected with pCMV-HBeAg at different concentrations for 24 h. SOCS1, SOCS2, and SOCS3 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( B – E ) HepG2 cells were incubated with rHBeAg at different concentrations ( B ) or transfected with pCMV-HBeAg for different times ( C ). HepG2 cells were transfected with pCMV-Tag2B, pCMV-HBeAg, or pCMV-HBeAg-1896mut for 24 h ( D ) or incubated with PBS, rHBeAg, heat-inactivated HBeAg, or rHBcAg for 12 h ( E ). SOCS2 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( F ) HepG2, pBlue-SK-transfected HepG2, pHBV1.3-transfected HepG2, or HepG2.2.15 cells were harvested 24 h after transfection. SOCS2 mRNAs were measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( G ) HepG2 cells were transfected with pBlue-SK or pHBV1.3 for different times. SOCS2 mRNA was measured by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( H ) HepG2 cells were transfected with pCMV-tag2B or plasmids expressing individual HBV proteins for 48 h. SOCS2 mRNA was determined by real-time PCR (upper). SOCS2 and β-actin mRNAs were determined by semi-quantitative RT-PCR (lower). ( I ) HepG2 cells were transfected with pBlue-SK, pHBV1.3, or pHBV1.3-1896mut for 24 h. SOCS2 mRNA was determined by real-time PCR (upper). SOCS2 and β-actin proteins were detected by Western blot analyses (lower). ( J ) HepG2 cells were pretreated with each of specific kinase inhibitors for 12 h, and incubated with rHBeAg or PBS for 24 h. SOCS2 mRNAs were measured by real-time PCR. ( K ) HepG2 cells were incubated with PBS, rHBeAg, or 12-O-tetradecanoylphorbol 13-acetate (TPA) for 12 h. p-ERK, ERK, and GAPDH proteins were detected by Western blot analyses. Data shown are means ± SE; n = 3. *p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Quantitative RT-PCR

    A proposed mechanism by which HBV hijacks IFN/JAK/STAT pathway via activating SOCS2 to facilitate immune evasion and viral infection. During hepatitis B virus (HBV) infection, the extracellular viral protein (hepatitis B e antigen, HBeAg) activates the cellular factor (suppressor of cytokine signaling 2, SOCS2) expression through regulating the extracellular regulated protein kinase (ERK) signaling. Activated SOCS2 subsequently hijacks the IFN/JAK/STAT pathway to reduce tyrosine kinase 2 (TYK2) stability and phosphorylation, downregulate interferon-α/β receptor 1 (IFN-α/βR1, IFNAR1) and interferon-λ1 receptor (cytokine receptor family 2 member 4, CRF2-4 or IL-10Rβ) production, attenuate signal transducer and activator of transcription 1 (STAT1) phosphorylation and nucleus translocation, and finally block IFN-stimulated genes (ISGs) expression, which results in the facilitation of HBV immune evasion, persistent infection, and possible pathogenesis.
    Figure Legend Snippet: A proposed mechanism by which HBV hijacks IFN/JAK/STAT pathway via activating SOCS2 to facilitate immune evasion and viral infection. During hepatitis B virus (HBV) infection, the extracellular viral protein (hepatitis B e antigen, HBeAg) activates the cellular factor (suppressor of cytokine signaling 2, SOCS2) expression through regulating the extracellular regulated protein kinase (ERK) signaling. Activated SOCS2 subsequently hijacks the IFN/JAK/STAT pathway to reduce tyrosine kinase 2 (TYK2) stability and phosphorylation, downregulate interferon-α/β receptor 1 (IFN-α/βR1, IFNAR1) and interferon-λ1 receptor (cytokine receptor family 2 member 4, CRF2-4 or IL-10Rβ) production, attenuate signal transducer and activator of transcription 1 (STAT1) phosphorylation and nucleus translocation, and finally block IFN-stimulated genes (ISGs) expression, which results in the facilitation of HBV immune evasion, persistent infection, and possible pathogenesis.

    Techniques Used: Infection, Expressing, Translocation Assay, Blocking Assay

    30) Product Images from "Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer"

    Article Title: Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19273

    ERK and p38 signaling pathways are involved in periostin-promoted EMT in lung cancer cells (A) A549 cells were incubated with periostin (100 ng/ml) for the indicated times, and phosphorylation of EKR, p38 and JNK was determined by Western Blot analysis. (B and C) A549 and CL1-0 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min and then incubated with periostin (100 ng/ml) for 24 h, and expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (D) A549 cells were treated as described in (B), and scattering morphology was photographed. (E-H) A549 and CL1-0 cells were treated as described in (B), and the wound healing assay, migration and invasion assays were assessed. (I and J) A549 and CL1-0 cells were transfected with ERK, p38 and JNK siRNA for 24 h, and incubated with periostin (100 ng/ml) for 24 h, while expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (K-O) A549 and CL1-0 cells were treated as described in (I) , and scattering morphology, the wound healing assay, migration and invasion assays were assessed. Results are expressed as the mean ± S.E.M. *p
    Figure Legend Snippet: ERK and p38 signaling pathways are involved in periostin-promoted EMT in lung cancer cells (A) A549 cells were incubated with periostin (100 ng/ml) for the indicated times, and phosphorylation of EKR, p38 and JNK was determined by Western Blot analysis. (B and C) A549 and CL1-0 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min and then incubated with periostin (100 ng/ml) for 24 h, and expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (D) A549 cells were treated as described in (B), and scattering morphology was photographed. (E-H) A549 and CL1-0 cells were treated as described in (B), and the wound healing assay, migration and invasion assays were assessed. (I and J) A549 and CL1-0 cells were transfected with ERK, p38 and JNK siRNA for 24 h, and incubated with periostin (100 ng/ml) for 24 h, while expression levels of E-cadherin, N-cadherin, vimentin, Snail and Twist were examined by qRT-PCR. (K-O) A549 and CL1-0 cells were treated as described in (I) , and scattering morphology, the wound healing assay, migration and invasion assays were assessed. Results are expressed as the mean ± S.E.M. *p

    Techniques Used: Incubation, Western Blot, Expressing, Quantitative RT-PCR, Wound Healing Assay, Migration, Transfection

    Periostin induces Twist and Snail expression by miR-381 repression in lung cancer cells (A) Schematic selection of candidate miRNAs. miR-381, which targets Twist and Snail 3’ UTR regions, was selected by using 3 online computational algorithms TargetScan, miRWalk and miRanda. (B) A549 cells were incubated with periostin (0-100 ng/ml) for 24 h. Total RNA was extracted and miR-381 expression was assessed by qRT-PCR. (C-D) A549 cells were transfected with control microRNA or miR-381 mimic for 24 h, then incubated with periostin for 24 h. Total RNA or protein was extracted, and Twist and Snail expression was assessed by qRT-PCR and Western Blot analysis. (E-G) A549 cells were transfected with miR-381 mimic or control mimic for 24 h, and incubated with periostin (100 ng/ml) for 24 h, and assessed by the wound healing assay, migration and invasion assays. (H and I) A549 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min or transfected with ERK, p38 and JNK siRNA for 24 h, then incubated with periostin (100 ng/ml) for 24 h. miR-381 expression was assessed by qRT-PCR. Results are expressed as the mean ± S.E.M. *p
    Figure Legend Snippet: Periostin induces Twist and Snail expression by miR-381 repression in lung cancer cells (A) Schematic selection of candidate miRNAs. miR-381, which targets Twist and Snail 3’ UTR regions, was selected by using 3 online computational algorithms TargetScan, miRWalk and miRanda. (B) A549 cells were incubated with periostin (0-100 ng/ml) for 24 h. Total RNA was extracted and miR-381 expression was assessed by qRT-PCR. (C-D) A549 cells were transfected with control microRNA or miR-381 mimic for 24 h, then incubated with periostin for 24 h. Total RNA or protein was extracted, and Twist and Snail expression was assessed by qRT-PCR and Western Blot analysis. (E-G) A549 cells were transfected with miR-381 mimic or control mimic for 24 h, and incubated with periostin (100 ng/ml) for 24 h, and assessed by the wound healing assay, migration and invasion assays. (H and I) A549 cells were pretreated with U0126 (10 μM), SB203580 (10 μM), or SP600125 (10 μM) for 30 min or transfected with ERK, p38 and JNK siRNA for 24 h, then incubated with periostin (100 ng/ml) for 24 h. miR-381 expression was assessed by qRT-PCR. Results are expressed as the mean ± S.E.M. *p

    Techniques Used: Expressing, Selection, Incubation, Quantitative RT-PCR, Transfection, Western Blot, Wound Healing Assay, Migration

    31) Product Images from "A Central Bioactive Region of LTBP-2 Stimulates the Expression of TGF-β1 in Fibroblasts via Akt and p38 Signalling Pathways"

    Article Title: A Central Bioactive Region of LTBP-2 Stimulates the Expression of TGF-β1 in Fibroblasts via Akt and p38 Signalling Pathways

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18102114

    Exogenous LTBP-2 stimulates phosphorylation of AKT and p38 mitogen-activated protein kinase (MAPK) in human fibroblasts. ( A ) MSU-1.1 cells (1 × 10 5 cells/well) were treated with or without LTBP-2 (10 µg/mL) for 30 min. Total cell lysates were immunoblotted for phosphorylation of candidate signalling molecules, including phospho-serine, phospho-tyrosine, phospho-threonine, phospho-p38 MAPK, phospho-Akt1/2/3, phospho-ERK, c-FOS, c-JUN, and for β-actin internal control as described in materials and methods. Note that there was major phosphorylation of p38 MAPK and AKT1/2/3, but no stimulation of ERK or cFOS; ( B ) Cells were treated for 30 min with full-length LTBP-2 or with molar equivalents of fragments containing TGF-β1 stimulating activity, LTBP-2C(H), or LTBP-2C(H) F3. Cell lysates were immunoblotted for total and phosphorylated p38 MAPK, Akt1/2/3, and ERK; ( C ) The ratio of phospho-protein to total protein for each signal molecule from each treatment is expressed relative to the average value from no LTBP-2 control cells (given an arbitrary value of 1.0). Similar results were observed in replicate experiments.
    Figure Legend Snippet: Exogenous LTBP-2 stimulates phosphorylation of AKT and p38 mitogen-activated protein kinase (MAPK) in human fibroblasts. ( A ) MSU-1.1 cells (1 × 10 5 cells/well) were treated with or without LTBP-2 (10 µg/mL) for 30 min. Total cell lysates were immunoblotted for phosphorylation of candidate signalling molecules, including phospho-serine, phospho-tyrosine, phospho-threonine, phospho-p38 MAPK, phospho-Akt1/2/3, phospho-ERK, c-FOS, c-JUN, and for β-actin internal control as described in materials and methods. Note that there was major phosphorylation of p38 MAPK and AKT1/2/3, but no stimulation of ERK or cFOS; ( B ) Cells were treated for 30 min with full-length LTBP-2 or with molar equivalents of fragments containing TGF-β1 stimulating activity, LTBP-2C(H), or LTBP-2C(H) F3. Cell lysates were immunoblotted for total and phosphorylated p38 MAPK, Akt1/2/3, and ERK; ( C ) The ratio of phospho-protein to total protein for each signal molecule from each treatment is expressed relative to the average value from no LTBP-2 control cells (given an arbitrary value of 1.0). Similar results were observed in replicate experiments.

    Techniques Used: Activity Assay

    32) Product Images from "Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway"

    Article Title: Factors released from embryonic stem cells inhibit apoptosis in H9c2 cells through PI3K/Akt but not ERK pathway

    Journal:

    doi: 10.1152/ajpheart.00279.2008

    Effects of ES-CM, Akt inhibitor LY-294002 (LY, 40 μM) and ERK inhibitor PD-98050 (PD, 25 μM) on H9c2 cell survival and proliferation after exposure to H 2 O 2 . A : quantitative number of H9c2 cells. Data are from the set of 7–9 independent
    Figure Legend Snippet: Effects of ES-CM, Akt inhibitor LY-294002 (LY, 40 μM) and ERK inhibitor PD-98050 (PD, 25 μM) on H9c2 cell survival and proliferation after exposure to H 2 O 2 . A : quantitative number of H9c2 cells. Data are from the set of 7–9 independent

    Techniques Used:

    Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on phosphorylated (p)Akt and pERK activity of H9c2 cells after exposure to H 2 O 2 . A : quantitative pAkt examined by case ELISA kit. Data are from the set of 5 to 6
    Figure Legend Snippet: Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on phosphorylated (p)Akt and pERK activity of H9c2 cells after exposure to H 2 O 2 . A : quantitative pAkt examined by case ELISA kit. Data are from the set of 5 to 6

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

    Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on TUNEL-positive nuclei ( A ), caspase-3 activity ( B ), and cell-death ELISA ( C and D ) after exposure to H 2 O 2 . A : LY, but not PD, inhibits ES-CM-reduced apoptosis,
    Figure Legend Snippet: Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on TUNEL-positive nuclei ( A ), caspase-3 activity ( B ), and cell-death ELISA ( C and D ) after exposure to H 2 O 2 . A : LY, but not PD, inhibits ES-CM-reduced apoptosis,

    Techniques Used: TUNEL Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on H9c2 cells apoptosis after exposure to H 2 O 2 . Representative photomicrographs of total nuclei stained with 4′,6-diamidino-2-phenylindole in blue ( A –
    Figure Legend Snippet: Effects of ES-CM, Akt inhibitor LY (40 μM), and ERK inhibitor PD (25 μM) on H9c2 cells apoptosis after exposure to H 2 O 2 . Representative photomicrographs of total nuclei stained with 4′,6-diamidino-2-phenylindole in blue ( A –

    Techniques Used: Staining

    33) Product Images from "RNAi-mediated Downregulation of Urokinase Plasminogen Activator Receptor (uPAR) and Matrix Metalloprotease-9 (MMP-9) in Human Breast Cancer Cells Results in Decreased Tumor Invasion, Angiogenesis and Growth"

    Article Title: RNAi-mediated Downregulation of Urokinase Plasminogen Activator Receptor (uPAR) and Matrix Metalloprotease-9 (MMP-9) in Human Breast Cancer Cells Results in Decreased Tumor Invasion, Angiogenesis and Growth

    Journal:

    doi: 10.1002/ijc.22962

    siRNA against uPAR and MMP-9 Inhibits the Levels of Phosphorylated ERK, MAPK, and AKT
    Figure Legend Snippet: siRNA against uPAR and MMP-9 Inhibits the Levels of Phosphorylated ERK, MAPK, and AKT

    Techniques Used:

    34) Product Images from "Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival"

    Article Title: Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076941

    IRE1 localizes at the MAM. (a) The subcellular distribution of ER stress sensors. All endogenous proteins were prepared from wild-type non-stressed CHO cells. P1, nuclear; Mito, mitochondrial; P3, microsomal; Cyt, cytosolic fractions. NucleoP, nucleoporin p62; Complex V, complex V ATP synthase inhibitor; Cyto c, cytochrome c; ERp57, ER thiol-disulfide oxidoreductase p57; ERK, extracellular signal-regulated kinase. Phosphatidylserine (PtSer) synthase activity was measured by the autoradiographic measurement of 14 C-PtSer as described [10] . All other proteins were measured by immunoblotting. (b) The subcellular distribution of IRE1 in CHO cells with reduced expression of mitofusin-2 (MFN2) or Sig-1Rs. Control (siCon) or specific siRNAs (siMFN2, siSig-1R) were transfected two days before the membrane fractionation. (c) Confocal microscopic observation of the subcellular distribution of Sig-1Rs and IRE1 in CHO cells. In top panels, endogenous ERp57 and transfected full-length IRE1-V5 were immunostained. Asterisks indicate CHO cells transfected with IRE1-V5 (Note: no V5 immunoreactivity in non-transfected cells, verifying the high selectivity of V5 immunostaining). In bottom panels, GFP targeting mitochondria was expressed by gene transfection. Arrows indicate clusters of IRE1-FLAG apposing mitochondria. Scale = 10 µm. Insets on a 5× magnification.
    Figure Legend Snippet: IRE1 localizes at the MAM. (a) The subcellular distribution of ER stress sensors. All endogenous proteins were prepared from wild-type non-stressed CHO cells. P1, nuclear; Mito, mitochondrial; P3, microsomal; Cyt, cytosolic fractions. NucleoP, nucleoporin p62; Complex V, complex V ATP synthase inhibitor; Cyto c, cytochrome c; ERp57, ER thiol-disulfide oxidoreductase p57; ERK, extracellular signal-regulated kinase. Phosphatidylserine (PtSer) synthase activity was measured by the autoradiographic measurement of 14 C-PtSer as described [10] . All other proteins were measured by immunoblotting. (b) The subcellular distribution of IRE1 in CHO cells with reduced expression of mitofusin-2 (MFN2) or Sig-1Rs. Control (siCon) or specific siRNAs (siMFN2, siSig-1R) were transfected two days before the membrane fractionation. (c) Confocal microscopic observation of the subcellular distribution of Sig-1Rs and IRE1 in CHO cells. In top panels, endogenous ERp57 and transfected full-length IRE1-V5 were immunostained. Asterisks indicate CHO cells transfected with IRE1-V5 (Note: no V5 immunoreactivity in non-transfected cells, verifying the high selectivity of V5 immunostaining). In bottom panels, GFP targeting mitochondria was expressed by gene transfection. Arrows indicate clusters of IRE1-FLAG apposing mitochondria. Scale = 10 µm. Insets on a 5× magnification.

    Techniques Used: Activity Assay, Expressing, Transfection, Fractionation, Immunostaining

    Sigma-1 receptors stabilize IRE1. (a) Sig-1R knockdown decreases phosphorylated IRE1 (pIRE1) in various types of cells when cells are under ER stress. Two days after the transfection of control siRNA (siCon) or Sig-1R siRNA (siSig-1R), cells were treated with thapsigargin (Tg) at 1 µM for 60 min. IRE1 were immunoprecipitated from 60–1000 µg of total protein lysates. (b) The temporal course of pIRE1 levels during ER stress. Control or Sig-1R siRNA was transfected to CHO cells two days before Tg. pIRE1 was measured by immunoprecipitation. The level of pIRE1 (partially phosphorylated IRE1 plus hyperphosphorylated IRE1) was normalized to ERK. The graph represents the means±S.E.M. ***p
    Figure Legend Snippet: Sigma-1 receptors stabilize IRE1. (a) Sig-1R knockdown decreases phosphorylated IRE1 (pIRE1) in various types of cells when cells are under ER stress. Two days after the transfection of control siRNA (siCon) or Sig-1R siRNA (siSig-1R), cells were treated with thapsigargin (Tg) at 1 µM for 60 min. IRE1 were immunoprecipitated from 60–1000 µg of total protein lysates. (b) The temporal course of pIRE1 levels during ER stress. Control or Sig-1R siRNA was transfected to CHO cells two days before Tg. pIRE1 was measured by immunoprecipitation. The level of pIRE1 (partially phosphorylated IRE1 plus hyperphosphorylated IRE1) was normalized to ERK. The graph represents the means±S.E.M. ***p

    Techniques Used: Transfection, Immunoprecipitation

    35) Product Images from "Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status"

    Article Title: Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093335

    Differential role of PI3K/Akt and MAPK/ERK pathways in conjugate induced apoptosis of prostate cancer cells. Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at p
    Figure Legend Snippet: Differential role of PI3K/Akt and MAPK/ERK pathways in conjugate induced apoptosis of prostate cancer cells. Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at p

    Techniques Used: Transfection, Concentration Assay

    Proposed scheme for conjugate mediated actions on LNCaP and PC-3 prostate cancer cells. Conjugate inhibits both androgen receptor and ERK signalling and finally contributing to its downstream effects of decreased cell viability and increased apoptosis in androgen receptor positive LNCaP cells. In case of androgen receptor negative PC-3 cells, inhibition of Akt and its downstream targets contributes to conjugate mediated decrease in cell viability and increased apoptosis.
    Figure Legend Snippet: Proposed scheme for conjugate mediated actions on LNCaP and PC-3 prostate cancer cells. Conjugate inhibits both androgen receptor and ERK signalling and finally contributing to its downstream effects of decreased cell viability and increased apoptosis in androgen receptor positive LNCaP cells. In case of androgen receptor negative PC-3 cells, inhibition of Akt and its downstream targets contributes to conjugate mediated decrease in cell viability and increased apoptosis.

    Techniques Used: Inhibition

    Differential role of PI3K/Akt and MAPK/ERK pathways in conjugate induced apoptosis of prostate cancer cells. Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at p
    Figure Legend Snippet: Differential role of PI3K/Akt and MAPK/ERK pathways in conjugate induced apoptosis of prostate cancer cells. Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at p

    Techniques Used: Transfection, Concentration Assay

    36) Product Images from "Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins"

    Article Title: Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins

    Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

    doi: 10.1096/fj.07-099697

    H 2 O 2 stimulates EGFR tyrosine phosphorylation. A , B ) T 84 monolayers were bilaterally treated with a range of concentrations of H 2 O 2 for 5 min ( A ; n =6) or with 500 μM H 2 O 2 over different time points ( B ; n =6). EGFR immunoprecipitates were probed for tyrosine phosphorylation levels by Western blot analysis. Bands were quantified by densitometric analysis. C, D ) Monolayers were preincubated with either the EGFR kinase inhibitor, tyrphostin AG1478 (1 μM), or a neutralizing antibody against the ligand-binding domain of the EGFR (5 μg/ml) to inhibit EGFR activation prior to treatment with H 2 O 2 . H 2 O 2 -induced EGFR ( C ; n =6), and ERK phosphorylation ( D ; n =6) was determined by Western blot analysis and densitometry. E ) T 84 monolayers were preincubated with tyrphostin AG1478 (1 μM), and H 2 O 2 -induced p38 phosphorylation was determined by Western blot analysis and densitometry (histogram; n =3). F ) T 84 monolayers mounted in Ussing chambers were pretreated with tyrphostin AG1478 (1 μM) prior to addition of H 2 O 2 , and subsequent I sc responses to CCh (100 μM) were measured ( n =6). Results are presented as means ± SE for levels of phosphorylation (a.u., arbitrary units). * P
    Figure Legend Snippet: H 2 O 2 stimulates EGFR tyrosine phosphorylation. A , B ) T 84 monolayers were bilaterally treated with a range of concentrations of H 2 O 2 for 5 min ( A ; n =6) or with 500 μM H 2 O 2 over different time points ( B ; n =6). EGFR immunoprecipitates were probed for tyrosine phosphorylation levels by Western blot analysis. Bands were quantified by densitometric analysis. C, D ) Monolayers were preincubated with either the EGFR kinase inhibitor, tyrphostin AG1478 (1 μM), or a neutralizing antibody against the ligand-binding domain of the EGFR (5 μg/ml) to inhibit EGFR activation prior to treatment with H 2 O 2 . H 2 O 2 -induced EGFR ( C ; n =6), and ERK phosphorylation ( D ; n =6) was determined by Western blot analysis and densitometry. E ) T 84 monolayers were preincubated with tyrphostin AG1478 (1 μM), and H 2 O 2 -induced p38 phosphorylation was determined by Western blot analysis and densitometry (histogram; n =3). F ) T 84 monolayers mounted in Ussing chambers were pretreated with tyrphostin AG1478 (1 μM) prior to addition of H 2 O 2 , and subsequent I sc responses to CCh (100 μM) were measured ( n =6). Results are presented as means ± SE for levels of phosphorylation (a.u., arbitrary units). * P

    Techniques Used: Western Blot, Ligand Binding Assay, Activation Assay

    37) Product Images from "Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo"

    Article Title: Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.01.008

    Effect of rociletinib on AKT, ERK, and their phosphorylations in MDR and the parental cells. S1 and S1-MI-80 cells were treated with different concentrations of rociletinib for 48 h. The protein expression level of AKT and ERK and phosphorylations were detected by Western blot (GAPDH as loading control). All these experiments were repeated at least three times.
    Figure Legend Snippet: Effect of rociletinib on AKT, ERK, and their phosphorylations in MDR and the parental cells. S1 and S1-MI-80 cells were treated with different concentrations of rociletinib for 48 h. The protein expression level of AKT and ERK and phosphorylations were detected by Western blot (GAPDH as loading control). All these experiments were repeated at least three times.

    Techniques Used: Expressing, Western Blot

    38) Product Images from "Indirubin 3′-Oxime Inhibits Migration, Invasion, and Metastasis InVivo in Mice Bearing Spontaneously Occurring Pancreatic Cancer via Blocking the RAF/ERK, AKT, and SAPK/JNK Pathways"

    Article Title: Indirubin 3′-Oxime Inhibits Migration, Invasion, and Metastasis InVivo in Mice Bearing Spontaneously Occurring Pancreatic Cancer via Blocking the RAF/ERK, AKT, and SAPK/JNK Pathways

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2019.08.010

    Inhibition of phosphorylated RAF/ERK, AKT, SAPK/JNK and c-Jun by Indox. ( A ) After treatment with indicated concentration of Indox for 24 h, low levels of p-B-RAF (Ser446), p-ERK (Tyr204), p-AKT (Thr308), p-SAPK/JNK (Tyr183) and p-c-Jun (Thr91) in the murine PDAC cell line (#146) were determined by immunoblotting. ( B ) Quantification of data presented in A . * P
    Figure Legend Snippet: Inhibition of phosphorylated RAF/ERK, AKT, SAPK/JNK and c-Jun by Indox. ( A ) After treatment with indicated concentration of Indox for 24 h, low levels of p-B-RAF (Ser446), p-ERK (Tyr204), p-AKT (Thr308), p-SAPK/JNK (Tyr183) and p-c-Jun (Thr91) in the murine PDAC cell line (#146) were determined by immunoblotting. ( B ) Quantification of data presented in A . * P

    Techniques Used: Inhibition, Concentration Assay

    Effects of Indox on the levels of phosphorylated RAF/ERK, AKT, SAPK/JNK pathways. Antibody array revealed that phosphorylation of RAF/ERK ( A ), AKTs ( B ), MAP/MMK ( C ), SAPK/JNK ( D ), and c-Jun ( E ) in the murine cell line (#146) was inhibited by 10 μM Indox for 24 h (n = 6). * P
    Figure Legend Snippet: Effects of Indox on the levels of phosphorylated RAF/ERK, AKT, SAPK/JNK pathways. Antibody array revealed that phosphorylation of RAF/ERK ( A ), AKTs ( B ), MAP/MMK ( C ), SAPK/JNK ( D ), and c-Jun ( E ) in the murine cell line (#146) was inhibited by 10 μM Indox for 24 h (n = 6). * P

    Techniques Used: Ab Array

    39) Product Images from "Root Bark of Paeonia suffruticosa Extract and Its Component Methyl Gallate Possess Peroxynitrite Scavenging Activity and Anti-Inflammatory Properties through NF-κB Inhibition in LPS-treated Mice"

    Article Title: Root Bark of Paeonia suffruticosa Extract and Its Component Methyl Gallate Possess Peroxynitrite Scavenging Activity and Anti-Inflammatory Properties through NF-κB Inhibition in LPS-treated Mice

    Journal: Molecules

    doi: 10.3390/molecules24193483

    Total and phosphorylated p-ERK, p-JNK, and p-p38 protein levels in the kidney. N, normal mice; Veh, vehicle-administered and LPS-treated mice; PM20, P. suffruticosa 20 mg/kg body weight-administered and LPS-treated mice; PM100, P. suffruticosa 100 mg/kg body weight-administered and LPS-treated mice; and MG5, methyl gallate 5 mg/kg body weight-administered and LPS-treated mice. The protein levels of p-ERK, p-JNK, and p-p38 were quantified using CS analyzer software. Respective total-form proteins were used as loading control. A representation of three experiments that yielded similar results. One-factor ANOVA: # p
    Figure Legend Snippet: Total and phosphorylated p-ERK, p-JNK, and p-p38 protein levels in the kidney. N, normal mice; Veh, vehicle-administered and LPS-treated mice; PM20, P. suffruticosa 20 mg/kg body weight-administered and LPS-treated mice; PM100, P. suffruticosa 100 mg/kg body weight-administered and LPS-treated mice; and MG5, methyl gallate 5 mg/kg body weight-administered and LPS-treated mice. The protein levels of p-ERK, p-JNK, and p-p38 were quantified using CS analyzer software. Respective total-form proteins were used as loading control. A representation of three experiments that yielded similar results. One-factor ANOVA: # p

    Techniques Used: Mouse Assay, Software

    40) Product Images from "Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance"

    Article Title: Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2020.102699

    High intracellular serine contributes to ERK phosphorylation. A. Volcano plot showing the topmost 20 differentially expressed metabolic genes in the microarray of Gln-deprived HLE cells, 24 h. Genes having - log2FC are downregulated; +log2FC are upregulated. B. Schematic depiction of the serine biosynthesis pathway, including the interconnection between serine, glycine and methionine; on the right, intracellular metabolite data showing serine (SER), glycine (GLY) and methionine (MET) levels in Gln-deprived HUH7 and HLE cells. Error bars indicate mean ± S .E.M, representative of 3 experiments. C. Crystal violet staining comparing the effect of 5 mM serine, glycine and methionine on HLE cells, 72 h. Here the cells were seeded at a higher density prior to the metabolite supplementation. Representative of n > 2 experiments. D. Clonogenic assay showing the selective effect of serine load on HLE cells. Representative of n > 2 experiments, 72 h. Cell number in – Gln condition was 2x CM condition. E. CellTitre Glo viability assay following serine deprivation in a panel of HCC cell lines, after 48 h. The error bars indicate mean ± SD, n = 4. F. Intracellular metabolic profiling showing the level of serine after 24 h culture in the indicated culture media conditions. The error bars indicate mean ± SD, n = 3 samples. G. Intracellular metabolic profiling showing the level of glutamine after 24 h in the indicated culture media conditions. The error bars indicate mean ± SD, n = 3 samples. H. Extracellular level of serine and glutamine as well as intracellular glutamine after 24 h in the indicated culture media conditions. Error bars indicate mean ± SD, n = 3 samples. I. Western blot showing the effect of serine load on pERK levels compared to glycine and methionine, 24 h. Each of the three metabolites were supplemented at 10 mM concentration. J. Western blot densitometric plots of the effect of serine removal or supplementation on pERK levels in 5 HCC cell lines, 24 h. Representative of 2 technical repeat experiments. K. Clonogenic assay showing the protective effect of blocking the ERK pathway on HLE cells supplemented with serine in Gln-deprived state. Cells were treated for 3 days in the indicated conditions, followed by culture in complete media for another 4 days. DMSO – dimethyl sulphoxide (drug solvent).
    Figure Legend Snippet: High intracellular serine contributes to ERK phosphorylation. A. Volcano plot showing the topmost 20 differentially expressed metabolic genes in the microarray of Gln-deprived HLE cells, 24 h. Genes having - log2FC are downregulated; +log2FC are upregulated. B. Schematic depiction of the serine biosynthesis pathway, including the interconnection between serine, glycine and methionine; on the right, intracellular metabolite data showing serine (SER), glycine (GLY) and methionine (MET) levels in Gln-deprived HUH7 and HLE cells. Error bars indicate mean ± S .E.M, representative of 3 experiments. C. Crystal violet staining comparing the effect of 5 mM serine, glycine and methionine on HLE cells, 72 h. Here the cells were seeded at a higher density prior to the metabolite supplementation. Representative of n > 2 experiments. D. Clonogenic assay showing the selective effect of serine load on HLE cells. Representative of n > 2 experiments, 72 h. Cell number in – Gln condition was 2x CM condition. E. CellTitre Glo viability assay following serine deprivation in a panel of HCC cell lines, after 48 h. The error bars indicate mean ± SD, n = 4. F. Intracellular metabolic profiling showing the level of serine after 24 h culture in the indicated culture media conditions. The error bars indicate mean ± SD, n = 3 samples. G. Intracellular metabolic profiling showing the level of glutamine after 24 h in the indicated culture media conditions. The error bars indicate mean ± SD, n = 3 samples. H. Extracellular level of serine and glutamine as well as intracellular glutamine after 24 h in the indicated culture media conditions. Error bars indicate mean ± SD, n = 3 samples. I. Western blot showing the effect of serine load on pERK levels compared to glycine and methionine, 24 h. Each of the three metabolites were supplemented at 10 mM concentration. J. Western blot densitometric plots of the effect of serine removal or supplementation on pERK levels in 5 HCC cell lines, 24 h. Representative of 2 technical repeat experiments. K. Clonogenic assay showing the protective effect of blocking the ERK pathway on HLE cells supplemented with serine in Gln-deprived state. Cells were treated for 3 days in the indicated conditions, followed by culture in complete media for another 4 days. DMSO – dimethyl sulphoxide (drug solvent).

    Techniques Used: Microarray, Staining, Clonogenic Assay, Viability Assay, Western Blot, Concentration Assay, Blocking Assay

    Blocking the ERK pathway in an impaired metabolic state enables proliferation. A. MTT proliferation assay following treatment of HUH7 and HLE cells with Sorafenib (multi-kinase inhibitor) Erlotinib (EGFR inhibitor), and MEK/pERK inhibitor U0126, in complete medium, 48 h. The asterisks indicate significant p-value compared to the untreated group. B. Western blot analysis of HUH7 and HLE cells treated with the indicated inhibitors, 24 h. C. Proliferation assay, showing the effect of Sorafenib, Erlotinib and U0126 in Gln-deprived media, 48 h. On the right, effect of U0126 in media containing Asparaginase. Asterisks indicate significant p-value compared to the untreated group (UT), which refers to cells cultured in – Gln or in Asparaginase media without U0126. D. Clonogenic assay showing the response of Gln-deprived HUH7 and HLE cells to the indicated MEK inhibitors. The inhibitor concentrations are as follows: U0126 (10 μM), SCH772984 (0.25 μM), Trametinib (0.5 μM). Cells were treated for 4 days, followed by rescue with complete media for 3 days. E. Pathway annotation of 569 genes that were upregulated in Gln-deprived HLE, but suppressed by U0126 treatment [20 µM]. The annotation profile was generated with DAVID ( https://david.ncifcrf.gov/tools.jsp ). The embedded Venn diagram shows the total number of differentially expressed genes at P
    Figure Legend Snippet: Blocking the ERK pathway in an impaired metabolic state enables proliferation. A. MTT proliferation assay following treatment of HUH7 and HLE cells with Sorafenib (multi-kinase inhibitor) Erlotinib (EGFR inhibitor), and MEK/pERK inhibitor U0126, in complete medium, 48 h. The asterisks indicate significant p-value compared to the untreated group. B. Western blot analysis of HUH7 and HLE cells treated with the indicated inhibitors, 24 h. C. Proliferation assay, showing the effect of Sorafenib, Erlotinib and U0126 in Gln-deprived media, 48 h. On the right, effect of U0126 in media containing Asparaginase. Asterisks indicate significant p-value compared to the untreated group (UT), which refers to cells cultured in – Gln or in Asparaginase media without U0126. D. Clonogenic assay showing the response of Gln-deprived HUH7 and HLE cells to the indicated MEK inhibitors. The inhibitor concentrations are as follows: U0126 (10 μM), SCH772984 (0.25 μM), Trametinib (0.5 μM). Cells were treated for 4 days, followed by rescue with complete media for 3 days. E. Pathway annotation of 569 genes that were upregulated in Gln-deprived HLE, but suppressed by U0126 treatment [20 µM]. The annotation profile was generated with DAVID ( https://david.ncifcrf.gov/tools.jsp ). The embedded Venn diagram shows the total number of differentially expressed genes at P

    Techniques Used: Blocking Assay, MTT Assay, Proliferation Assay, Western Blot, Cell Culture, Clonogenic Assay, Generated

    Related Articles

    Western Blot:

    Article Title: REGULATION OF NEURONAL PLCγ BY CHRONIC MORPHINE
    Article Snippet: .. The following antibodies were used for western blotting studies: ERK1, ERK2, phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA); PLCγ1 (Upstate Biotechnology, Inc., Lake Placid, NY); GFP (Clontech, Palo Alto, CA); phospho-ERK (1:10,000, Promega, Madison, WI); and phospho-PLCγ 1 (1:2000) was generated for these studies as described below. .. Peroxidase-linked goat anti-rabbit and rabbit anti-mouse antibodies, used at a 1:2000 dilution, were obtained from Vector Labs (Burlingame, CA).

    Generated:

    Article Title: REGULATION OF NEURONAL PLCγ BY CHRONIC MORPHINE
    Article Snippet: .. The following antibodies were used for western blotting studies: ERK1, ERK2, phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA); PLCγ1 (Upstate Biotechnology, Inc., Lake Placid, NY); GFP (Clontech, Palo Alto, CA); phospho-ERK (1:10,000, Promega, Madison, WI); and phospho-PLCγ 1 (1:2000) was generated for these studies as described below. .. Peroxidase-linked goat anti-rabbit and rabbit anti-mouse antibodies, used at a 1:2000 dilution, were obtained from Vector Labs (Burlingame, CA).

    other:

    Article Title: Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer
    Article Snippet: Filters were probed with the indicated primary antibodies: anti-PDGFRβ, anti-phospho-44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2, indicated as p-ERK1/2), anti-phospho-Akt (Ser473, indicated as p-Akt), anti-Akt (Cell Signaling Technology Inc.), anti-PD-L1/CD274 (Proteintech Group, Inc.), anti-ERK1 (C-16) and anti-vinculin (N-19) (Santa Cruz Biotechnology, Santa Cruz, CA).

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    Santa Cruz Biotechnology erk
    TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against <t>ERK</t> as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against <t>Rictor</t> as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.
    Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti erk ab
    Proposed mechanism of the combined action of FTS + MPA on EC cells Estrogens play an important role in the regulation of cell proliferation, differentiation, and function of the endometrium. They mediate their biological effects through two estrogen receptors in the nucleus, namely ERα and ERβ. ERs are transcription factors that are activated by phosphorylation and control gene expression. The activation is induced either in response to ligand binding or independently of ligand. Ras becomes active in the Ras-GTP form, which is upregulated by extracellular signals such as growth factors and hormones that bind the tyrosine kinase receptor. Once activated, Ras-GTP signals to multiple effector pathways that regulate proliferation, survival, metabolism, migration and shape of the cell. One of these pathways causes phosphorylation of <t>Akt,</t> which leads to cell survival, and another causes phosphorylation of <t>ERK,</t> leading to cell proliferation. Nuclear ERα is phosphorylated by pAkt at Ser167 and by pERK at Ser-118. Once phosphorylated, ERα is activated to target the transcription of genes (such as c-fos, ps2/TFF1 , and PR A+B ), leading to cell survival and proliferation. MPA increases ERα degradation, thereby reducing ERα in the cell (both in the nucleus and in the cytoplasm). FTS inhibits active Ras-GTP, leading to a decrease in pAkt and pERK, and hence a decrease in pERαSer118 and in pERαSer167, and finally a decrease in ERα gene transcription. These results showed that the combination of MPA + FTS inhibits proliferation of endometrial cells. ERα, estrogen receptor alpha; FTS, S-farnesylthiosalicylic acid; MPA, medroxyprogesterone acetate; PI3K, phosphatidylinositide 3-kinase; s118, serine-118; s167, serine-167.
    Rabbit Anti Erk Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology p42 erk 2
    (A) Syk −/− macrophages display reduced phosphorylation of <t>p42</t> <t>Erk-2</t> after FcγR engagement. Activation of Erk-2 MAPK was visualized by the phosphorylation-induced mobility shift. Macrophages were allowed to bind to IgG-RBC, and after the indicated incubation times, cells were lysed and tested for the presence of doubly threonine-tyrosine-phosphorylated forms of Erk-2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (B) Induction of NOS2 (iNOS) by LPS-gamma interferon is normal in Syk-deficient macrophages. Macrophage cultures were induced with 10-μg/ml LPS and 2-U/ml gamma interferon for 16 h. At the end of the incubation period, cells were lysed and probed for the presence of NOS2 by Western blotting. WT, wild type.
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    TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.

    Journal: Molecular cancer research : MCR

    Article Title: Differential IKK/NF-κB Activity is Mediated by TSC2 through mTORC1 in PTEN-null Prostate Cancer and Tuberous Sclerosis Complex Tumor Cells

    doi: 10.1158/1541-7786.MCR-15-0213

    Figure Lengend Snippet: TSC2 promotes NF-κB signaling in TSC2 deficient cells through mTORC1 inhibition-induced activation of PI3K/Akt. A. The hypothesis of TSC2 regulation of IKK/NF-κB in TSC2 deficient cells. B. EEF126-8 (Tsc2−/−) cells were transfected with TSC2 (wild type), lysed, and analyzed by western blots. C. Cells were transfected with siRNA control or siRNA against Akt1 plus Akt2 as indicated, lysed and analyzed by western blots. D. Cells were transfected with siRNA control or siRNA against ERK as indicated. Cell lysates were analyzed by western blots. E. Cells were transfected with siRNA control or siRNA against Rictor as indicated, lysed and analyzed by western blots. F. Cells were transfected with siRNA control or siRNA against Raptor, lysed and analyzed by western blots. These experiments were repeated for three times.

    Article Snippet: Each of these represents four pooled SMART-selected siRNA duplexes that target the indicated mRNA. siRNA to human TSC2-(2) was from Cell Signaling Technology (CST-6476). siRNA to human Raptor was from Dharmacon. siRNA to rat Akt1, Akt2, Raptor, Rictor and ERK and siRNA to mouse Rictor, Raptor, Akt1, Akt2 and ERK were from Santa Cruz.

    Techniques: Inhibition, Activation Assay, Transfection, Western Blot

    Effects of the combination of Rg1 and TA3 on signaling molecules in MG63 human osteosarcoma cells. (a) Activation of JNK, ERK, and p38, which are the representative MAPKs, is synergistically attenuated by Rg1 and TA3. (b) β -catenin and active form of CREB (p-CREB) were synergistically suppressed by Rg1 and TA3.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Ginsenoside Rg1 Drives Stimulations of Timosaponin AIII-Induced Anticancer Effects in Human Osteosarcoma Cells

    doi: 10.1155/2020/8980124

    Figure Lengend Snippet: Effects of the combination of Rg1 and TA3 on signaling molecules in MG63 human osteosarcoma cells. (a) Activation of JNK, ERK, and p38, which are the representative MAPKs, is synergistically attenuated by Rg1 and TA3. (b) β -catenin and active form of CREB (p-CREB) were synergistically suppressed by Rg1 and TA3.

    Article Snippet: Primary antibodies against phospho-ERK (sc-7383), ERK (sc-94), JNK (sc-571), phospho-JNK (sc-6254), phospho-p38 (sc-17852-R), p38 (sc-7972), phospho-CREB (sc-7978-R), actin (sc-1615), and β -catenin (sc-7199) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Activation Assay

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

    Journal: Oncotarget

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

    doi:

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

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

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

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

    Journal: Oncotarget

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

    doi:

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

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

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

    (A) Syk −/− macrophages display reduced phosphorylation of p42 Erk-2 after FcγR engagement. Activation of Erk-2 MAPK was visualized by the phosphorylation-induced mobility shift. Macrophages were allowed to bind to IgG-RBC, and after the indicated incubation times, cells were lysed and tested for the presence of doubly threonine-tyrosine-phosphorylated forms of Erk-2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (B) Induction of NOS2 (iNOS) by LPS-gamma interferon is normal in Syk-deficient macrophages. Macrophage cultures were induced with 10-μg/ml LPS and 2-U/ml gamma interferon for 16 h. At the end of the incubation period, cells were lysed and probed for the presence of NOS2 by Western blotting. WT, wild type.

    Journal: Molecular and Cellular Biology

    Article Title: The Syk Protein Tyrosine Kinase Is Essential for Fc? Receptor Signaling in Macrophages and Neutrophils

    doi:

    Figure Lengend Snippet: (A) Syk −/− macrophages display reduced phosphorylation of p42 Erk-2 after FcγR engagement. Activation of Erk-2 MAPK was visualized by the phosphorylation-induced mobility shift. Macrophages were allowed to bind to IgG-RBC, and after the indicated incubation times, cells were lysed and tested for the presence of doubly threonine-tyrosine-phosphorylated forms of Erk-2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (B) Induction of NOS2 (iNOS) by LPS-gamma interferon is normal in Syk-deficient macrophages. Macrophage cultures were induced with 10-μg/ml LPS and 2-U/ml gamma interferon for 16 h. At the end of the incubation period, cells were lysed and probed for the presence of NOS2 by Western blotting. WT, wild type.

    Article Snippet: For Western blotting, we used antibodies against phosphotyrosine RC20 (Transduction Laboratories), Syk (generously provided by Bruce Rowley), the FcγR γ chain (generously provided by Marie-Helene Jouvin), p42 Erk-2 (Upstate Biochemicals), and NOS2 (Santa Cruz Biotechnology, Santa Cruz, Calif.).

    Techniques: Activation Assay, Mobility Shift, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot