phosphorylated mek1  (Cell Signaling Technology Inc)

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

    Cell Signaling Technology Inc phosphorylated mek1
    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and <t>MEK1</t> in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    The PathScan Total Smad2 3 Sandwich ELISA Kit is a solid phase sandwich enzyme linked immunosorbent assay ELISA that recognizes endogenous levels of Smad2 and Smad3 proteins A Smad2 3 Mouse Antibody has been coated on the microwells After incubation with cell lysates Smad2 3 proteins phospho and nonphospho are captured by the coated antibody Following extensive washing a Smad2 3 Rabbit Detection Antibody is added to detect captured Smad2 3 proteins Anti rabbit IgG HRP linked Antibody is then used to recognize the bound detection antibody HRP substrate TMB is added to develop color The magnitude of the absorbance for this developed color is proportional to the quantity of Smad2 and Smad3 proteins
    https://www.bioz.com/result/phosphorylated mek1/product/Cell Signaling Technology Inc
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    Images

    1) Product Images from "Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling"

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Journal: Molecules

    doi: 10.3390/molecules21030301

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    Figure Legend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    2) Product Images from "CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation"

    Article Title: CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002135

    Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P
    Figure Legend Snippet: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P

    Techniques Used: Western Blot

    3) Product Images from "Atrial ERK1/2 activation in the embryo leads to incomplete Septal closure: a novel mouse model of atrial Septal defect"

    Article Title: Atrial ERK1/2 activation in the embryo leads to incomplete Septal closure: a novel mouse model of atrial Septal defect

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-017-0392-2

    Regulation of the MEK-ERK signaling pathway in 14.5 dpc hearts from two different aMEK1 transgenic models. Despite evidence of aMEK1 expression in the atria of both TAMEK/MH DTg and MEK1 single Tg mice, increased ERK1/2 phosphorylation was documented only in the DTg. * P
    Figure Legend Snippet: Regulation of the MEK-ERK signaling pathway in 14.5 dpc hearts from two different aMEK1 transgenic models. Despite evidence of aMEK1 expression in the atria of both TAMEK/MH DTg and MEK1 single Tg mice, increased ERK1/2 phosphorylation was documented only in the DTg. * P

    Techniques Used: Transgenic Assay, Expressing, Mouse Assay

    Regulation of MEK1 transgene and ERK1/2 expression. a Activation of the HA-tagged human aMEK1 is regulated by the expression of tTA and doxycycline (DOX) in the TAMEK/MH DTg mouse heart. b At 12.5 dpc, fetal hearts expressed the aMEK1 transgene when the pregnant females did not receive DOX in their drinking water. Expression of aMEK1 transgene was absent in the fetal brain. c At 14.5 dpc, the expression of aMEK1 transgene. And corresponding ERK1/2 phosphorylation, is higher in the atria than in the ventricles as demonstrated in paired tissues ( n = 5). d Quantitation of MEK1/2, ERK1/2 and P-ERK1/2 protein from water- (W) or DOX- (D) treated mice at 12.5 dpc, and from ventricular (V) and atrial (A) tissue at 14.5 dpc. Original blots available in Additional file 5 S5
    Figure Legend Snippet: Regulation of MEK1 transgene and ERK1/2 expression. a Activation of the HA-tagged human aMEK1 is regulated by the expression of tTA and doxycycline (DOX) in the TAMEK/MH DTg mouse heart. b At 12.5 dpc, fetal hearts expressed the aMEK1 transgene when the pregnant females did not receive DOX in their drinking water. Expression of aMEK1 transgene was absent in the fetal brain. c At 14.5 dpc, the expression of aMEK1 transgene. And corresponding ERK1/2 phosphorylation, is higher in the atria than in the ventricles as demonstrated in paired tissues ( n = 5). d Quantitation of MEK1/2, ERK1/2 and P-ERK1/2 protein from water- (W) or DOX- (D) treated mice at 12.5 dpc, and from ventricular (V) and atrial (A) tissue at 14.5 dpc. Original blots available in Additional file 5 S5

    Techniques Used: Expressing, Activation Assay, Quantitation Assay, Mouse Assay

    Proposed role of baseline MKP3 expression in determining ERK1/2 activation and, in turn, putative induction of ASD phenotype in transgenic mice with expression of constitutively active MEK1 (see Discussion)
    Figure Legend Snippet: Proposed role of baseline MKP3 expression in determining ERK1/2 activation and, in turn, putative induction of ASD phenotype in transgenic mice with expression of constitutively active MEK1 (see Discussion)

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

    4) Product Images from "MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways"

    Article Title: MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

    Journal: Marine Drugs

    doi: 10.3390/md13127071

    Effects of MytiLec treatment on MEK, ERK, and cell cycle-related molecules in Burkitt’s lymphoma Ramos cells. ( A ) Phosphorylation and expression levels of MEK1/2, ERK1/2 and p21, p27, CDK6 and cyclinD3 were shown, respectively. Cells (4 × 10 5 in each experiment) were treated with various concentrations of MytiLec as shown, and activation levels were evaluated by Western blotting of lysates. Solid and dotted lines indicated increasing and decreasing trends, respectively. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ( B ) Relative densitometric quantification of P-MEK/MEK, P-ERK/ERK and p21/GAPDH. Each experiment was repeated three times.
    Figure Legend Snippet: Effects of MytiLec treatment on MEK, ERK, and cell cycle-related molecules in Burkitt’s lymphoma Ramos cells. ( A ) Phosphorylation and expression levels of MEK1/2, ERK1/2 and p21, p27, CDK6 and cyclinD3 were shown, respectively. Cells (4 × 10 5 in each experiment) were treated with various concentrations of MytiLec as shown, and activation levels were evaluated by Western blotting of lysates. Solid and dotted lines indicated increasing and decreasing trends, respectively. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ( B ) Relative densitometric quantification of P-MEK/MEK, P-ERK/ERK and p21/GAPDH. Each experiment was repeated three times.

    Techniques Used: Expressing, Activation Assay, Western Blot

    5) Product Images from "Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line"

    Article Title: Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2008.05409.x

    4HPR-induced phosphorylation of ERK1/2 is blocked by U0126 Panel A, 4HPR-induced phosphorylation of ERK1/2 is time-dependent. ARPE-19 cells in culture were treated with 1 μM of 4HPR for indicated time points, and the cell extracts were analyzed for phosphorylation of ERK1/2 by Western blotting described under Materials and Methods. Panel B , the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. Cells were pretreated with 1 μM of U0126 for 1 h followed by 4HPR for additional 48 h, then analyzed by Western blotting. Panel C , ELISA analysis of the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. The cultured ARPE-19 cells were treated with 1 μM of 4HPR for 48 h in the presence or absence of U0126, and the phosphorylation of p44/42 MAPK was measured by PathScan®Phospho-p44/42 MAPK sandwich ELISA kit as described under Materials and Methods. The values are mean ± SD, n = 4. * P
    Figure Legend Snippet: 4HPR-induced phosphorylation of ERK1/2 is blocked by U0126 Panel A, 4HPR-induced phosphorylation of ERK1/2 is time-dependent. ARPE-19 cells in culture were treated with 1 μM of 4HPR for indicated time points, and the cell extracts were analyzed for phosphorylation of ERK1/2 by Western blotting described under Materials and Methods. Panel B , the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. Cells were pretreated with 1 μM of U0126 for 1 h followed by 4HPR for additional 48 h, then analyzed by Western blotting. Panel C , ELISA analysis of the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. The cultured ARPE-19 cells were treated with 1 μM of 4HPR for 48 h in the presence or absence of U0126, and the phosphorylation of p44/42 MAPK was measured by PathScan®Phospho-p44/42 MAPK sandwich ELISA kit as described under Materials and Methods. The values are mean ± SD, n = 4. * P

    Techniques Used: Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Cell Culture, Sandwich ELISA

    6) Product Images from "Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line"

    Article Title: Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2008.05409.x

    Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.
    Figure Legend Snippet: Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.

    Techniques Used: Activation Assay, Marker

    MEK1/2 pathway mediates 4HPR-induced neuronal differentiation of ARPE-19 cells Cultured ARPE-19 cells were pretreated with 1 μM U0126 for 1 h followed by incubation with 1μM 4HPR for additional 72 h. Cell lysates were prepared, and then analyzed by Western blotting using non-phospho or phospho-specific antibodies of MAPK/ERK pathway as described under Materials and Methods. Panel A, 4HPR-induced phosphorylation of c-Raf and MEK1/2 were not blocked by U0126. Panel B , the inhibition of 4HPR-induced phosphorylation of p90RSK and c-Fos by U0126. Panel C, the inhibition of 4HPR-induced phosphorylation of SAPK/JNK and c-Jun by U0126.
    Figure Legend Snippet: MEK1/2 pathway mediates 4HPR-induced neuronal differentiation of ARPE-19 cells Cultured ARPE-19 cells were pretreated with 1 μM U0126 for 1 h followed by incubation with 1μM 4HPR for additional 72 h. Cell lysates were prepared, and then analyzed by Western blotting using non-phospho or phospho-specific antibodies of MAPK/ERK pathway as described under Materials and Methods. Panel A, 4HPR-induced phosphorylation of c-Raf and MEK1/2 were not blocked by U0126. Panel B , the inhibition of 4HPR-induced phosphorylation of p90RSK and c-Fos by U0126. Panel C, the inhibition of 4HPR-induced phosphorylation of SAPK/JNK and c-Jun by U0126.

    Techniques Used: Cell Culture, Incubation, Western Blot, Inhibition

    7) Product Images from "Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A"

    Article Title: Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-015-0226-1

    Effects of palmitoylation on signaling transduction of oncogenic NRAS vs. KRAS4A. Serum-starved lysates of NIH3T3 cells expressing GFP, KRAS4A G12D , KRAS4A G12D/C180S , NRAS G12D , or NRAS G12D/C181S were analyzed by western blotting. a Phospho-c-RAF, MEK, and ERK1/2 antibodies were used to detect the effects of palmitoylation deficiency on the MEK-ERK signaling pathway. b Phospho-AKT and S6RP antibodies were used to detect the effects of palmitoylation deficiency on the PI3K-AKT signaling pathway. Expression levels of RAS, GFP, and β-Actin were detected as loading controls
    Figure Legend Snippet: Effects of palmitoylation on signaling transduction of oncogenic NRAS vs. KRAS4A. Serum-starved lysates of NIH3T3 cells expressing GFP, KRAS4A G12D , KRAS4A G12D/C180S , NRAS G12D , or NRAS G12D/C181S were analyzed by western blotting. a Phospho-c-RAF, MEK, and ERK1/2 antibodies were used to detect the effects of palmitoylation deficiency on the MEK-ERK signaling pathway. b Phospho-AKT and S6RP antibodies were used to detect the effects of palmitoylation deficiency on the PI3K-AKT signaling pathway. Expression levels of RAS, GFP, and β-Actin were detected as loading controls

    Techniques Used: Transduction, Expressing, Western Blot

    8) Product Images from "Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A"

    Article Title: Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-015-0226-1

    Effects of palmitoylation on signaling transduction of oncogenic NRAS vs. KRAS4A. Serum-starved lysates of NIH3T3 cells expressing GFP, KRAS4A G12D , KRAS4A G12D/C180S , NRAS G12D , or NRAS G12D/C181S were analyzed by western blotting. a Phospho-c-RAF, MEK, and ERK1/2 antibodies were used to detect the effects of palmitoylation deficiency on the MEK-ERK signaling pathway. b Phospho-AKT and S6RP antibodies were used to detect the effects of palmitoylation deficiency on the PI3K-AKT signaling pathway. Expression levels of RAS, GFP, and β-Actin were detected as loading controls
    Figure Legend Snippet: Effects of palmitoylation on signaling transduction of oncogenic NRAS vs. KRAS4A. Serum-starved lysates of NIH3T3 cells expressing GFP, KRAS4A G12D , KRAS4A G12D/C180S , NRAS G12D , or NRAS G12D/C181S were analyzed by western blotting. a Phospho-c-RAF, MEK, and ERK1/2 antibodies were used to detect the effects of palmitoylation deficiency on the MEK-ERK signaling pathway. b Phospho-AKT and S6RP antibodies were used to detect the effects of palmitoylation deficiency on the PI3K-AKT signaling pathway. Expression levels of RAS, GFP, and β-Actin were detected as loading controls

    Techniques Used: Transduction, Expressing, Western Blot

    9) Product Images from "Met provides essential signals for liver regeneration"

    Article Title: Met provides essential signals for liver regeneration

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

    doi: 10.1073/pnas.0403412101

    Activation of signaling cascades in the regenerating liver of conditional Met mutant mice. Western blot analysis of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, phospho-GSK3-β, total GSK3-β protein, phospho-STAT3, and total STAT3 protein in control and conditional Met mutant liver is shown at the indicated time points after partial hepatectomy.
    Figure Legend Snippet: Activation of signaling cascades in the regenerating liver of conditional Met mutant mice. Western blot analysis of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, phospho-GSK3-β, total GSK3-β protein, phospho-STAT3, and total STAT3 protein in control and conditional Met mutant liver is shown at the indicated time points after partial hepatectomy.

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

    10) Product Images from "Unraveling heterogeneous susceptibility and the evolution of breast cancer using a systems biology approach"

    Article Title: Unraveling heterogeneous susceptibility and the evolution of breast cancer using a systems biology approach

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0599-z

    Analysis of representative signaling molecules from pathways downstream of ERBB2 in the tumors. (A) Positive correlation between levels of pERK 1/2 (ELISA) and tumor latency. (B) Evaluation of upstream and downstream elements from the ERK 1/2 pathway in tumors with short and long latencies. Tumors with a short latency show low levels of pERK 1/2 and pMEK and lower levels of KSR 1/2 . (C) Evaluation of some elements of signaling pathways downstream of ERBB2 in F1BX tumors with and without lung metastases. Tumors that metastasize to the lung show low levels of pAKT(S473). We explored the AKT pathway to clarify this defect (Figure S6A in Additional file 2 ). This effect was not present in mice with a homogeneous genetic background (Figure S6B in Additional file 2 ). (D) The main pAKT isoform associated with dissemination to the lung in the F1BX population was pAKT1 (ELISA). (E) Breast tumors of human origin that disseminated show low levels of pAKT compared with those that did not metastasize.
    Figure Legend Snippet: Analysis of representative signaling molecules from pathways downstream of ERBB2 in the tumors. (A) Positive correlation between levels of pERK 1/2 (ELISA) and tumor latency. (B) Evaluation of upstream and downstream elements from the ERK 1/2 pathway in tumors with short and long latencies. Tumors with a short latency show low levels of pERK 1/2 and pMEK and lower levels of KSR 1/2 . (C) Evaluation of some elements of signaling pathways downstream of ERBB2 in F1BX tumors with and without lung metastases. Tumors that metastasize to the lung show low levels of pAKT(S473). We explored the AKT pathway to clarify this defect (Figure S6A in Additional file 2 ). This effect was not present in mice with a homogeneous genetic background (Figure S6B in Additional file 2 ). (D) The main pAKT isoform associated with dissemination to the lung in the F1BX population was pAKT1 (ELISA). (E) Breast tumors of human origin that disseminated show low levels of pAKT compared with those that did not metastasize.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    11) Product Images from "Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3"

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2008.08.007

    MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.
    Figure Legend Snippet: MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.

    Techniques Used: Transfection, Positive Control, Incubation

    TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.
    Figure Legend Snippet: TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.

    Techniques Used: Incubation

    Reciprocal regulation of ERK1/2 and MKP-3. Phosphorylation and activation of ERK1/2 typically occurs through stimulation of the Ras/Raf/MEK1/2 protein kinase cascade. The GTPase Ras activates the protein kinase Raf, which phosphorylates and activates the protein kinases MEK1/2. MEK1/2 is highly specific for phosphorylating and activating ERK1/2. The magnitude and duration of ERK1/2 activity is determined by the balance between the activity of MEK1/2, the kinases that phosphorylate and activate ERK1/2, and the activity of phosphatases, such as MKP-3, which dephosphorylate and inactivate ERK1/2. The levels of MKP-3 protein available to inhibit ERK1/2 are regulated, at least in part, by ERK1/2. ERK1/2 can stimulate an increase in the expression of MKP-3 RNA. This results in an increase in MKP-3 protein, which can then feedback and inhibit ERK1/2 activity. ERK1/2 can also stimulate a decrease in MKP-3 protein stability, however, which can cause a loss of MKP-3 protein. Thus, the levels of MKP-3 protein are determined, at least in part, by the balance between the stimulation of new MKP-3 gene expression and the stability of the MKP-3 protein.
    Figure Legend Snippet: Reciprocal regulation of ERK1/2 and MKP-3. Phosphorylation and activation of ERK1/2 typically occurs through stimulation of the Ras/Raf/MEK1/2 protein kinase cascade. The GTPase Ras activates the protein kinase Raf, which phosphorylates and activates the protein kinases MEK1/2. MEK1/2 is highly specific for phosphorylating and activating ERK1/2. The magnitude and duration of ERK1/2 activity is determined by the balance between the activity of MEK1/2, the kinases that phosphorylate and activate ERK1/2, and the activity of phosphatases, such as MKP-3, which dephosphorylate and inactivate ERK1/2. The levels of MKP-3 protein available to inhibit ERK1/2 are regulated, at least in part, by ERK1/2. ERK1/2 can stimulate an increase in the expression of MKP-3 RNA. This results in an increase in MKP-3 protein, which can then feedback and inhibit ERK1/2 activity. ERK1/2 can also stimulate a decrease in MKP-3 protein stability, however, which can cause a loss of MKP-3 protein. Thus, the levels of MKP-3 protein are determined, at least in part, by the balance between the stimulation of new MKP-3 gene expression and the stability of the MKP-3 protein.

    Techniques Used: Activation Assay, Activity Assay, Expressing

    12) Product Images from "β-amyloid cytotoxicity is prevented by natural achillolide A"

    Article Title: β-amyloid cytotoxicity is prevented by natural achillolide A

    Journal: Journal of Natural Medicines

    doi: 10.1007/s11418-018-1191-0

    Achillolide A attenuates the phosphorylation of p44/42 MAPK and SAPK/JNK induced by Aβ in N2a cells. Cells were either untreated or treated with Aβ only (25 μM) or Aβ + achillolide A for 30 min (p44/42 MAPK) or 40 min (SAPK/JNK). The levels of phosphorylated and total SAPK/JNK ( a ) and p44/42 MAPK ( b ) in cell extracts were determined by corresponding ELISA kits. The levels of the phosphorylated proteins were normalized to the levels of the total amount of the related proteins, and are presented as the mean ± SEM of two experiments ( n = 4) for SAPK/JNK, and three experiments ( n = 6) for p44/42 MAPK. The levels of the phosphorylated proteins were significantly lower in cells treated with both Aβ + achillolide A compared to cells treated with Aβ only. **p
    Figure Legend Snippet: Achillolide A attenuates the phosphorylation of p44/42 MAPK and SAPK/JNK induced by Aβ in N2a cells. Cells were either untreated or treated with Aβ only (25 μM) or Aβ + achillolide A for 30 min (p44/42 MAPK) or 40 min (SAPK/JNK). The levels of phosphorylated and total SAPK/JNK ( a ) and p44/42 MAPK ( b ) in cell extracts were determined by corresponding ELISA kits. The levels of the phosphorylated proteins were normalized to the levels of the total amount of the related proteins, and are presented as the mean ± SEM of two experiments ( n = 4) for SAPK/JNK, and three experiments ( n = 6) for p44/42 MAPK. The levels of the phosphorylated proteins were significantly lower in cells treated with both Aβ + achillolide A compared to cells treated with Aβ only. **p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    13) Product Images from "CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation"

    Article Title: CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002135

    Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P
    Figure Legend Snippet: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P

    Techniques Used: Western Blot

    Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Material and Methods . (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ 1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ 1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (* P
    Figure Legend Snippet: Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Material and Methods . (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ 1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ 1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (* P

    Techniques Used: Inhibition, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

    14) Product Images from "Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line"

    Article Title: Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2008.05409.x

    4HPR-induced phosphorylation of ERK1/2 is blocked by U0126 Panel A, 4HPR-induced phosphorylation of ERK1/2 is time-dependent. ARPE-19 cells in culture were treated with 1 μM of 4HPR for indicated time points, and the cell extracts were analyzed for phosphorylation of ERK1/2 by Western blotting described under Materials and Methods. Panel B , the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. Cells were pretreated with 1 μM of U0126 for 1 h followed by 4HPR for additional 48 h, then analyzed by Western blotting. Panel C , ELISA analysis of the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. The cultured ARPE-19 cells were treated with 1 μM of 4HPR for 48 h in the presence or absence of U0126, and the phosphorylation of p44/42 MAPK was measured by PathScan®Phospho-p44/42 MAPK sandwich ELISA kit as described under Materials and Methods. The values are mean ± SD, n = 4. * P
    Figure Legend Snippet: 4HPR-induced phosphorylation of ERK1/2 is blocked by U0126 Panel A, 4HPR-induced phosphorylation of ERK1/2 is time-dependent. ARPE-19 cells in culture were treated with 1 μM of 4HPR for indicated time points, and the cell extracts were analyzed for phosphorylation of ERK1/2 by Western blotting described under Materials and Methods. Panel B , the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. Cells were pretreated with 1 μM of U0126 for 1 h followed by 4HPR for additional 48 h, then analyzed by Western blotting. Panel C , ELISA analysis of the inhibition of 4HPR-induced phosphorylation of ERK1/2 by U0126. The cultured ARPE-19 cells were treated with 1 μM of 4HPR for 48 h in the presence or absence of U0126, and the phosphorylation of p44/42 MAPK was measured by PathScan®Phospho-p44/42 MAPK sandwich ELISA kit as described under Materials and Methods. The values are mean ± SD, n = 4. * P

    Techniques Used: Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Cell Culture, Sandwich ELISA

    4HPR-induced neuronal differentiation is blocked by ERK1/2 siRNA Panel A, ERK1/2 siRNA blocked the neuronal differentiation induced by 4HPR. Cultured ARPE-19 cells were transfected with ERK1/2 siRNA, and after 24 h of post-transfection the cells were treated with 1 μM 4HPR for 72 h, and then analyzed by phase contrast microscopy as described under Materials and Methods. Scale bar = 100 μm. Panel B, ERK1/2 siRNA blocked the ERK1/2 phosphorylation induced by 4HPR. After 24 h of post-transfection with ERK1/2 or mock siRNA, the cells were treated with 1 μM of 4HPR for 72 h. Cell lysates were analyzed for phosphorylation of ERK1/2 by Western blotting. Panel C, ERK1/2 siRNA attenuated the expression of calretinin induced by 4HPR. Cells transfected with ERK1/2 or mock siRNA were treated with 1 μM 4HPR for 72 h after 24 h of post-transfection. Total RNA was extracted and analyzed by real time quantitative PCR as described under Materials and Methods. The values are mean ± SD, n = 4. * P
    Figure Legend Snippet: 4HPR-induced neuronal differentiation is blocked by ERK1/2 siRNA Panel A, ERK1/2 siRNA blocked the neuronal differentiation induced by 4HPR. Cultured ARPE-19 cells were transfected with ERK1/2 siRNA, and after 24 h of post-transfection the cells were treated with 1 μM 4HPR for 72 h, and then analyzed by phase contrast microscopy as described under Materials and Methods. Scale bar = 100 μm. Panel B, ERK1/2 siRNA blocked the ERK1/2 phosphorylation induced by 4HPR. After 24 h of post-transfection with ERK1/2 or mock siRNA, the cells were treated with 1 μM of 4HPR for 72 h. Cell lysates were analyzed for phosphorylation of ERK1/2 by Western blotting. Panel C, ERK1/2 siRNA attenuated the expression of calretinin induced by 4HPR. Cells transfected with ERK1/2 or mock siRNA were treated with 1 μM 4HPR for 72 h after 24 h of post-transfection. Total RNA was extracted and analyzed by real time quantitative PCR as described under Materials and Methods. The values are mean ± SD, n = 4. * P

    Techniques Used: Cell Culture, Transfection, Microscopy, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.
    Figure Legend Snippet: Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.

    Techniques Used: Activation Assay, Marker

    15) Product Images from "CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation"

    Article Title: CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002135

    Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P
    Figure Legend Snippet: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P

    Techniques Used: Western Blot

    Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Material and Methods . (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ 1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ 1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (* P
    Figure Legend Snippet: Inhibition of both p38 and/or p44/42 pathways further enhances CD45RB cross-linking mediated microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Material and Methods . (A) Cell supernatants and lysates were analyzed for extracellular (top panel) and cell-associated (bottom panel) FITC-Aβ 1–42 using a fluorometer. Data are represented as the relative fold of mean fluorescence change (mean±SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (n = 6 for each condition presented). Cell lysates (B, C) were assayed for microglial phagocytosis of Aβ 1–42 peptide by Aβ-ELISA. Results are reported as picogram per microgram of total protein for cells incubated at 37°C over cells incubated at 4°C. (37°C/4°C; n = 3 for each condition presented). One-way ANOVA followed by post hoc Bonferroni testing revealed significant between-group differences (* P

    Techniques Used: Inhibition, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

    16) Product Images from "p53 suppresses carcinoma progression by inhibiting mTOR pathway activation"

    Article Title: p53 suppresses carcinoma progression by inhibiting mTOR pathway activation

    Journal: Oncogene

    doi: 10.1038/onc.2013.589

    p53 loss results in mTOR pathway activation in Rb deficient MTC Activated, phosphorylated forms of S6 kinase (p-S6K), AKT (p-AKT) and MEK (p-MEK1/2) as well as corresponding total protein levels (S6K, AKT and MEK-1) were compared between Rb and Rb/p53 ablated thyroid tumors by Western blot analysis. (a) p-S6K was significantly increased in Rb/p53 as compared to Rb ablated tumors despite similar total S6K levels. (b) p-AKT and total AKT levels were similar in Rb and Rb/p53 ablated tumors. (c) p-MEK was significantly decreased in Rb/p53 as compared to Rb ablated tumors despite similar total MEK levels. Quantification is represented as relative densitometric values of phosphorylated:total protein ratios. (d) p53 loss in Rb deficient MTC results in decreased expression of mTOR pathway inhibitors. Schematic diagram illustrating previously identified p53 target genes (blue outlined boxes) known to repress mTOR signaling. Activation is indicated in blue and suppression in red. Quantitative RT-PCR showed significantly reduced expression of Sesn2, Tsc2, Plk2, Igfbp3 and Pten (blue shaded boxes), but not Ddit4 and Prkab1 (unshaded boxes), in Rb/p53 as compared to Rb ablated MTC. Data are represented as mean ± SD. *p
    Figure Legend Snippet: p53 loss results in mTOR pathway activation in Rb deficient MTC Activated, phosphorylated forms of S6 kinase (p-S6K), AKT (p-AKT) and MEK (p-MEK1/2) as well as corresponding total protein levels (S6K, AKT and MEK-1) were compared between Rb and Rb/p53 ablated thyroid tumors by Western blot analysis. (a) p-S6K was significantly increased in Rb/p53 as compared to Rb ablated tumors despite similar total S6K levels. (b) p-AKT and total AKT levels were similar in Rb and Rb/p53 ablated tumors. (c) p-MEK was significantly decreased in Rb/p53 as compared to Rb ablated tumors despite similar total MEK levels. Quantification is represented as relative densitometric values of phosphorylated:total protein ratios. (d) p53 loss in Rb deficient MTC results in decreased expression of mTOR pathway inhibitors. Schematic diagram illustrating previously identified p53 target genes (blue outlined boxes) known to repress mTOR signaling. Activation is indicated in blue and suppression in red. Quantitative RT-PCR showed significantly reduced expression of Sesn2, Tsc2, Plk2, Igfbp3 and Pten (blue shaded boxes), but not Ddit4 and Prkab1 (unshaded boxes), in Rb/p53 as compared to Rb ablated MTC. Data are represented as mean ± SD. *p

    Techniques Used: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    17) Product Images from "Survival of pancreatic cancer cells lacking KRAS function"

    Article Title: Survival of pancreatic cancer cells lacking KRAS function

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00942-5

    PI3K inhibition functions through AKT-dependent and -independent mechanisms. a Western blot showed stable pERK1/2 but increased pAKT and pPRAS40 levels in 8988T and A13 KRAS deficient (purple) cells, consistent with PI3K/AKT pathway activation. HSP90 is loading control. b Dose-response curves of 8988T and A13 KRAS intact (gray) and deficient (purples) clones to the pan-AKT inhibitor MK2206. Each replicate ( n = 3 for each dose) and curve fit are shown. c Western blot showed sustained phosphorylation of AKT and downstream targets (PRAS40, S6, and 4EBP1) only in myr-AKT1- and myr-AKT2- expressing cells but not in myr-AKT1 (K179M)- or control GFP -expressing cells following 4 h of 2 μM GDC-0941 treatment. d Dose-response curves of cell lines in c treated with GDC-0941 and BAY80-6946 demonstrated a marked decrease in PI3K sensitivity with myr-AKT1 or myr-AKT2 overexpression. Each replicate ( n = 3 for each dose) and curve fit are shown
    Figure Legend Snippet: PI3K inhibition functions through AKT-dependent and -independent mechanisms. a Western blot showed stable pERK1/2 but increased pAKT and pPRAS40 levels in 8988T and A13 KRAS deficient (purple) cells, consistent with PI3K/AKT pathway activation. HSP90 is loading control. b Dose-response curves of 8988T and A13 KRAS intact (gray) and deficient (purples) clones to the pan-AKT inhibitor MK2206. Each replicate ( n = 3 for each dose) and curve fit are shown. c Western blot showed sustained phosphorylation of AKT and downstream targets (PRAS40, S6, and 4EBP1) only in myr-AKT1- and myr-AKT2- expressing cells but not in myr-AKT1 (K179M)- or control GFP -expressing cells following 4 h of 2 μM GDC-0941 treatment. d Dose-response curves of cell lines in c treated with GDC-0941 and BAY80-6946 demonstrated a marked decrease in PI3K sensitivity with myr-AKT1 or myr-AKT2 overexpression. Each replicate ( n = 3 for each dose) and curve fit are shown

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

    18) Product Images from "Met provides essential signals for liver regeneration"

    Article Title: Met provides essential signals for liver regeneration

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

    doi: 10.1073/pnas.0403412101

    Activation of signaling cascades in the regenerating liver of conditional Met mutant mice. Western blot analysis of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, phospho-GSK3-β, total GSK3-β protein, phospho-STAT3, and total STAT3 protein in control and conditional Met mutant liver is shown at the indicated time points after partial hepatectomy.
    Figure Legend Snippet: Activation of signaling cascades in the regenerating liver of conditional Met mutant mice. Western blot analysis of phosho-Erk1/2, total Erk1/2 protein, phospho-Akt and total Akt protein, phospho-GSK3-β, total GSK3-β protein, phospho-STAT3, and total STAT3 protein in control and conditional Met mutant liver is shown at the indicated time points after partial hepatectomy.

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

    19) Product Images from "Development of Highly Sensitive Biosensors of RAF Dimerization in Cells"

    Article Title: Development of Highly Sensitive Biosensors of RAF Dimerization in Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37213-2

    Effects of RAF inhibitors on the MAPK pathway in cancer cell lines. Phospho-MEK1/2, phospho-ERK1/2, and GAPDH levels in COLO 205 cells ( a – d ), AsPC-1 cells ( e – h ), A549 cells ( i – l ), A427 ( m – p ), and H-1975 cells ( q – t ) treated with dabrafenib, LY3009120, TAK-632, or PLX7904. Cells were treated with the indicated concentrations of inhibitors for 2 hours, and cell lysates were analysed by western blotting. Solid black lines represent the boundaries of images cropped from different blots. The membranes were cut out before blotting, and full-length blots are unavailable. Uncropped images are shown in Supplementary Fig. S5 .
    Figure Legend Snippet: Effects of RAF inhibitors on the MAPK pathway in cancer cell lines. Phospho-MEK1/2, phospho-ERK1/2, and GAPDH levels in COLO 205 cells ( a – d ), AsPC-1 cells ( e – h ), A549 cells ( i – l ), A427 ( m – p ), and H-1975 cells ( q – t ) treated with dabrafenib, LY3009120, TAK-632, or PLX7904. Cells were treated with the indicated concentrations of inhibitors for 2 hours, and cell lysates were analysed by western blotting. Solid black lines represent the boundaries of images cropped from different blots. The membranes were cut out before blotting, and full-length blots are unavailable. Uncropped images are shown in Supplementary Fig. S5 .

    Techniques Used: Western Blot

    20) Product Images from "Golgi stress–induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing"

    Article Title: Golgi stress–induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0418

    BFA treatment activates MEK1/2 and ERK1/2, but not p38 or JNK1/2 signaling. (A) 786-0 or (B) HeLa cells were treated with 40 nM BFA for the indicated duration. The indicated proteins were detected by Western blot. (C–E) 786-0 cells were treated for the indicated duration with 40 nM BFA (C), 1.75 µM GCA (D), or 500 nM MON in the presence or absence of the MEK inhibitor U0126, which was used at 10 μM. The specified proteins were detected by immunoblotting; cPARP: cleaved PARP. (A–E) Representative blots of one example out of three independent experiments are shown with the exception of E, where n = 1. (F) A549 cells were treated with 1.75 µM GCA in the presence or absence of the MEK inhibitor U0126 used at 10 μM for the indicated duration. (G) A549 cells stably expressing the indicated constructs were treated with 70 nM BFA in the presence or absence of 10 μM U0126 for 24 h. (H, I) ELK1 activity in response to 20-h treatment with the indicated concentrations of BFA (H) or GCA (I) in the absence or presence of 10 µM U0126 was measured using a dual-luciferase reporter assay. A representative example of two independent experiments (single compound treatment) is shown; the combinatorial treatment was done once. Three wells per condition were analyzed per experiment. A.U. indicates arbitrary units. Asterisks (*) represent significant differences between DMSO and treatment conditions: *, p
    Figure Legend Snippet: BFA treatment activates MEK1/2 and ERK1/2, but not p38 or JNK1/2 signaling. (A) 786-0 or (B) HeLa cells were treated with 40 nM BFA for the indicated duration. The indicated proteins were detected by Western blot. (C–E) 786-0 cells were treated for the indicated duration with 40 nM BFA (C), 1.75 µM GCA (D), or 500 nM MON in the presence or absence of the MEK inhibitor U0126, which was used at 10 μM. The specified proteins were detected by immunoblotting; cPARP: cleaved PARP. (A–E) Representative blots of one example out of three independent experiments are shown with the exception of E, where n = 1. (F) A549 cells were treated with 1.75 µM GCA in the presence or absence of the MEK inhibitor U0126 used at 10 μM for the indicated duration. (G) A549 cells stably expressing the indicated constructs were treated with 70 nM BFA in the presence or absence of 10 μM U0126 for 24 h. (H, I) ELK1 activity in response to 20-h treatment with the indicated concentrations of BFA (H) or GCA (I) in the absence or presence of 10 µM U0126 was measured using a dual-luciferase reporter assay. A representative example of two independent experiments (single compound treatment) is shown; the combinatorial treatment was done once. Three wells per condition were analyzed per experiment. A.U. indicates arbitrary units. Asterisks (*) represent significant differences between DMSO and treatment conditions: *, p

    Techniques Used: Western Blot, Stable Transfection, Expressing, Construct, Activity Assay, Luciferase, Reporter Assay

    21) Product Images from "Survival of pancreatic cancer cells lacking KRAS function"

    Article Title: Survival of pancreatic cancer cells lacking KRAS function

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00942-5

    PI3K inhibition functions through AKT-dependent and -independent mechanisms. a Western blot showed stable pERK1/2 but increased pAKT and pPRAS40 levels in 8988T and A13 KRAS deficient (purple) cells, consistent with PI3K/AKT pathway activation. HSP90
    Figure Legend Snippet: PI3K inhibition functions through AKT-dependent and -independent mechanisms. a Western blot showed stable pERK1/2 but increased pAKT and pPRAS40 levels in 8988T and A13 KRAS deficient (purple) cells, consistent with PI3K/AKT pathway activation. HSP90

    Techniques Used: Inhibition, Western Blot, Activation Assay

    22) Product Images from "CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation"

    Article Title: CD45RB Is a Novel Molecular Therapeutic Target to Inhibit A? Peptide-Induced Microglial MAPK Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002135

    Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P
    Figure Legend Snippet: Activated p38 MAPK and/or p44/42 MAPK by LPS negatively affects microglial phagocytosis of Aβ 1–42 peptide. Microglial treatment conditions are indicated and are further described in Materials and Method . Cell lysates were analyzed by Western immunoblotting using specific antibodies that recognize phosphorylated or total p38 MAPK and/or p44/42 MAPK at the indicated time points (A and B, top panel). Phosphorylation of both p38 MAPK and/or p44/42 MAPK after treatment with LPS was inhibited by SB203580 or PD98059 (A and B, bottom panel). Histograms below the immunoblots represent the mean band density ratio±1 SD (pp38 MAPK/total p38 MAPK and/or pp44/42 MAPK/total p44/42 MAPK; n = 3 for each condition presented; * P

    Techniques Used: Western Blot

    23) Product Images from "Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling"

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Journal: Molecules

    doi: 10.3390/molecules21030301

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    Figure Legend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    24) Product Images from "Soy protein inhibits inflammation-induced VCAM-1 and inflammatory cytokine induction by inhibiting the NF-κB and AKT signaling pathway in apolipoprotein E–deficient mice"

    Article Title: Soy protein inhibits inflammation-induced VCAM-1 and inflammatory cytokine induction by inhibiting the NF-κB and AKT signaling pathway in apolipoprotein E–deficient mice

    Journal: European journal of nutrition

    doi: 10.1007/s00394-013-0509-7

    SPI − inhibits LPS-induced AKT activation. ApoE–/– mice ( n = 4/diet) were fed CAS- or SPI − -diet for 1 week followed by LPS (20 μg/mouse) challenge for 5 h (experiment 5). Total lysates were prepared from liver ( a ) and aorta ( b ), and phosphorylation status of MAP kinase, and AKT were determined using PathScan multi-target sandwich ELISA kit (cell signaling) as described under section “Materials and methods.” Values are mean ± SD, n = 4. * P
    Figure Legend Snippet: SPI − inhibits LPS-induced AKT activation. ApoE–/– mice ( n = 4/diet) were fed CAS- or SPI − -diet for 1 week followed by LPS (20 μg/mouse) challenge for 5 h (experiment 5). Total lysates were prepared from liver ( a ) and aorta ( b ), and phosphorylation status of MAP kinase, and AKT were determined using PathScan multi-target sandwich ELISA kit (cell signaling) as described under section “Materials and methods.” Values are mean ± SD, n = 4. * P

    Techniques Used: Activation Assay, Mouse Assay, Sandwich ELISA

    25) Product Images from "Angiotensin Type 2 Receptor Signaling in Satellite Cells Potentiates Skeletal Muscle Regeneration *"

    Article Title: Angiotensin Type 2 Receptor Signaling in Satellite Cells Potentiates Skeletal Muscle Regeneration *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.585521

    AT2R knockdown inhibits myoblast differentiation. A , immunoblot of myoblast differentiation markers and MAPK signaling components in C2C12 cells after AT2R knockdown. C2C12 myoblasts were cultured in proliferation medium and transfected with AT2R or control siRNA. Two independent AT2R siRNAs were used, termed AT2R-a ( Aa ) and AT2R-b ( Ab ). After cells reached confluence (day 0), the medium was switched to differentiation medium, and cells were harvested after 1, 2, and 3 days. C , control. B , quantification of p-ERK1/2, p-p38MAPK, and p-JNK calculated from A . Data are presented as the ratio to total level of corresponding protein. C , cell proliferation, apoptosis, and viability in C2C12 cells. C2C12 myoblasts were transfected with control ( C ) or AT2R ( A ) siRNA, and cell proliferation, apoptosis, and viability were measured before and 1 day after induction of differentiation. Anti-notch antibody (N), staurosporine ( S ), and mitomycin C ( M ) were used as positive controls for cell proliferation, apoptosis, and cell viability, respectively. −, non-transfected control. Data are mean ± S.E. ( n = 3 and 8 in B and C , respectively. *, p
    Figure Legend Snippet: AT2R knockdown inhibits myoblast differentiation. A , immunoblot of myoblast differentiation markers and MAPK signaling components in C2C12 cells after AT2R knockdown. C2C12 myoblasts were cultured in proliferation medium and transfected with AT2R or control siRNA. Two independent AT2R siRNAs were used, termed AT2R-a ( Aa ) and AT2R-b ( Ab ). After cells reached confluence (day 0), the medium was switched to differentiation medium, and cells were harvested after 1, 2, and 3 days. C , control. B , quantification of p-ERK1/2, p-p38MAPK, and p-JNK calculated from A . Data are presented as the ratio to total level of corresponding protein. C , cell proliferation, apoptosis, and viability in C2C12 cells. C2C12 myoblasts were transfected with control ( C ) or AT2R ( A ) siRNA, and cell proliferation, apoptosis, and viability were measured before and 1 day after induction of differentiation. Anti-notch antibody (N), staurosporine ( S ), and mitomycin C ( M ) were used as positive controls for cell proliferation, apoptosis, and cell viability, respectively. −, non-transfected control. Data are mean ± S.E. ( n = 3 and 8 in B and C , respectively. *, p

    Techniques Used: Cell Culture, Transfection

    Proposed model for AT2R-mediated regulation of satellite cell differentiation. Initial cue(s) directing differentiation lead to rapid up-regulation of AT2R expression. During satellite cell differentiation, ERK1/2 phosphorylation is suppressed at the initial stage of differentiation, followed by an increase in the later stage ( dotted line ). AT2R inhibits ERK1/2 signaling at all stages of differentiation, resulting in activation of myogenic gene expression (myogenin and desmin) and myoblast differentiation. ERK1/2 inhibition increases AT2R expression, suggesting a positive feedback regulation. It is of note that it has been suggested that ERK1/2 positively regulates myotube formation in the later stage of differentiation, independently from the regulation of differentiation markers (data not shown; see “Discussion”).
    Figure Legend Snippet: Proposed model for AT2R-mediated regulation of satellite cell differentiation. Initial cue(s) directing differentiation lead to rapid up-regulation of AT2R expression. During satellite cell differentiation, ERK1/2 phosphorylation is suppressed at the initial stage of differentiation, followed by an increase in the later stage ( dotted line ). AT2R inhibits ERK1/2 signaling at all stages of differentiation, resulting in activation of myogenic gene expression (myogenin and desmin) and myoblast differentiation. ERK1/2 inhibition increases AT2R expression, suggesting a positive feedback regulation. It is of note that it has been suggested that ERK1/2 positively regulates myotube formation in the later stage of differentiation, independently from the regulation of differentiation markers (data not shown; see “Discussion”).

    Techniques Used: Cell Differentiation, Expressing, Activation Assay, Inhibition

    AT2R positively regulates myoblast differentiation via ERK1/2 signaling. A , immunoblot of myoblast differentiation markers of C2C12 cells. C2C12 myoblasts were cultured in proliferation medium, transfected with control ( C ) and AT2R ( A ) siRNA, and the medium switched to proliferation medium ± the ERK1/2 inhibitors U0126 and PD98059 ( PD ). After cells reached confluence, the medium was switched to differentiation medium, and cells were harvested after 24 h. DMSO , dimethyl sulfoxide. B , immunohistochemical staining of eMyHC in C2C12 myotubes. C2C12 myoblasts were cultured under the same condition as in A , and myotube formation was analyzed 2 days after differentiation. C , fusion index (calculated as the percentage of nuclei in eMyHC-positive myotubes divided by the total number of nuclei) of C2C12 myotubes calculated from B . Data are mean ± S.E. ( n = 5). **, p
    Figure Legend Snippet: AT2R positively regulates myoblast differentiation via ERK1/2 signaling. A , immunoblot of myoblast differentiation markers of C2C12 cells. C2C12 myoblasts were cultured in proliferation medium, transfected with control ( C ) and AT2R ( A ) siRNA, and the medium switched to proliferation medium ± the ERK1/2 inhibitors U0126 and PD98059 ( PD ). After cells reached confluence, the medium was switched to differentiation medium, and cells were harvested after 24 h. DMSO , dimethyl sulfoxide. B , immunohistochemical staining of eMyHC in C2C12 myotubes. C2C12 myoblasts were cultured under the same condition as in A , and myotube formation was analyzed 2 days after differentiation. C , fusion index (calculated as the percentage of nuclei in eMyHC-positive myotubes divided by the total number of nuclei) of C2C12 myotubes calculated from B . Data are mean ± S.E. ( n = 5). **, p

    Techniques Used: Cell Culture, Transfection, Immunohistochemistry, Staining

    26) Product Images from "Blueberry Opposes ?-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase"

    Article Title: Blueberry Opposes ?-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase

    Journal: Rejuvenation Research

    doi: 10.1089/rej.2008.0757

    BB suppresses microglial activation and enhances microglial phagocytosis of Aβ 1–42 peptide through a p44/42 MAPK-dependent pathway. Microglial treatment conditions are indicated and are further described in the Materials and Methods section.
    Figure Legend Snippet: BB suppresses microglial activation and enhances microglial phagocytosis of Aβ 1–42 peptide through a p44/42 MAPK-dependent pathway. Microglial treatment conditions are indicated and are further described in the Materials and Methods section.

    Techniques Used: Activation Assay

    27) Product Images from "Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells"

    Article Title: Effect of API-1 and FR180204 on cell proliferation and apoptosis in human DLD-1 and LoVo colorectal cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4995

    Effect of treatment with FR and API-1 alone or in combination on on intracellular signaling pathways in (A) DLD-1 and (B) LoVo cells. Cells were treated with the indicated concentrations of these agents for 24 and 48 h and protein levels of p-Akt, total Akt, p-ERK1/2 and total ERK1/2 were assayed by western blot analysis. The reference protein β-actin was used as an internal loading control. FR, FR180204; API-1, 4-amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide; Akt, v-akt murine thymoma viral oncogene homolog; p-Akt, phosphorylated Akt; ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated ERK1/2.
    Figure Legend Snippet: Effect of treatment with FR and API-1 alone or in combination on on intracellular signaling pathways in (A) DLD-1 and (B) LoVo cells. Cells were treated with the indicated concentrations of these agents for 24 and 48 h and protein levels of p-Akt, total Akt, p-ERK1/2 and total ERK1/2 were assayed by western blot analysis. The reference protein β-actin was used as an internal loading control. FR, FR180204; API-1, 4-amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-d]pyrimidine-6-carboxamide; Akt, v-akt murine thymoma viral oncogene homolog; p-Akt, phosphorylated Akt; ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated ERK1/2.

    Techniques Used: Western Blot

    28) Product Images from "Golgi stress–induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing"

    Article Title: Golgi stress–induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0418

    BFA treatment activates MEK1/2 and ERK1/2, but not p38 or JNK1/2 signaling. (A) 786-0 or (B) HeLa cells were treated with 40 nM BFA for the indicated duration. The indicated proteins were detected by Western blot. (C–E) 786-0 cells were treated for the indicated duration with 40 nM BFA (C), 1.75 µM GCA (D), or 500 nM MON in the presence or absence of the MEK inhibitor U0126, which was used at 10 μM. The specified proteins were detected by immunoblotting; cPARP: cleaved PARP. (A–E) Representative blots of one example out of three independent experiments are shown with the exception of E, where n = 1. (F) A549 cells were treated with 1.75 µM GCA in the presence or absence of the MEK inhibitor U0126 used at 10 μM for the indicated duration. (G) A549 cells stably expressing the indicated constructs were treated with 70 nM BFA in the presence or absence of 10 μM U0126 for 24 h. (H, I) ELK1 activity in response to 20-h treatment with the indicated concentrations of BFA (H) or GCA (I) in the absence or presence of 10 µM U0126 was measured using a dual-luciferase reporter assay. A representative example of two independent experiments (single compound treatment) is shown; the combinatorial treatment was done once. Three wells per condition were analyzed per experiment. A.U. indicates arbitrary units. Asterisks (*) represent significant differences between DMSO and treatment conditions: *, p
    Figure Legend Snippet: BFA treatment activates MEK1/2 and ERK1/2, but not p38 or JNK1/2 signaling. (A) 786-0 or (B) HeLa cells were treated with 40 nM BFA for the indicated duration. The indicated proteins were detected by Western blot. (C–E) 786-0 cells were treated for the indicated duration with 40 nM BFA (C), 1.75 µM GCA (D), or 500 nM MON in the presence or absence of the MEK inhibitor U0126, which was used at 10 μM. The specified proteins were detected by immunoblotting; cPARP: cleaved PARP. (A–E) Representative blots of one example out of three independent experiments are shown with the exception of E, where n = 1. (F) A549 cells were treated with 1.75 µM GCA in the presence or absence of the MEK inhibitor U0126 used at 10 μM for the indicated duration. (G) A549 cells stably expressing the indicated constructs were treated with 70 nM BFA in the presence or absence of 10 μM U0126 for 24 h. (H, I) ELK1 activity in response to 20-h treatment with the indicated concentrations of BFA (H) or GCA (I) in the absence or presence of 10 µM U0126 was measured using a dual-luciferase reporter assay. A representative example of two independent experiments (single compound treatment) is shown; the combinatorial treatment was done once. Three wells per condition were analyzed per experiment. A.U. indicates arbitrary units. Asterisks (*) represent significant differences between DMSO and treatment conditions: *, p

    Techniques Used: Western Blot, Stable Transfection, Expressing, Construct, Activity Assay, Luciferase, Reporter Assay

    29) Product Images from "Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD"

    Article Title: Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0144864

    Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in StHdh Q7/7 and StHdh Q111/111 cells at 0 mins and 10 mins of 100ng/ml EGF stimulation, either with or without a prior 2 hour incubation with 500nM AKT inhibitor VIII or 1μM MEK 1/2 inhibitor. In cases where inhibitors were not used, cells were incubated with the equivalent volume of DMSO for the same amount of time prior to treatment and processing. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. Grey asterisks indicate genotypic differences. N = 3 replications *p
    Figure Legend Snippet: Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in StHdh Q7/7 and StHdh Q111/111 cells at 0 mins and 10 mins of 100ng/ml EGF stimulation, either with or without a prior 2 hour incubation with 500nM AKT inhibitor VIII or 1μM MEK 1/2 inhibitor. In cases where inhibitors were not used, cells were incubated with the equivalent volume of DMSO for the same amount of time prior to treatment and processing. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. Grey asterisks indicate genotypic differences. N = 3 replications *p

    Techniques Used: Sandwich ELISA, Incubation

    Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in Hdh Q7/7 and Hdh Q111/111 primary cells at 0 mins and 10 mins of 100ng/ml EGF stimulation. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. N = 5 replications. *** p
    Figure Legend Snippet: Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in Hdh Q7/7 and Hdh Q111/111 primary cells at 0 mins and 10 mins of 100ng/ml EGF stimulation. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. N = 5 replications. *** p

    Techniques Used: Sandwich ELISA

    30) Product Images from "MEK2 Is Sufficient but Not Necessary for Proliferation and Anchorage-Independent Growth of SK-MEL-28 Melanoma Cells"

    Article Title: MEK2 Is Sufficient but Not Necessary for Proliferation and Anchorage-Independent Growth of SK-MEL-28 Melanoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017165

    Necessity of MEK1 and MEK2 signaling pathways for ERK activation in SK-MEL-28 cells. SK-MEL-28 cells were transfected with nothing (N), lipid only (L), non-silencing control siRNA (NS), MEK-specific siRNA (MEK1 siRNA 7, 8, 11, and MEK2 siRNA 9, and 14), pools of MEK-specific siRNA (MEK1 siRNA 7+8+11 and MEK2 siRNA 9+14) or combinations of pools of MEK1- and MEK2-specific siRNA (P1, MEK1 siRNA 8+11 plus MEK2 siRNA 9+14; P2, MEK1 siRNA 7+8+11 plus MEK2 siRNA 9+14) as described in Material and Methods . After transfection, cells were trypsinized and split into separate dishes for cell lysate collection and for cell cycle analysis (results shown in Table 1 ). Seventy-two hours later, whole cell lysates were harvested and immunoblotted. The efficiency of siRNA-mediated MEK knock down was examined by immunoblotting with antibodies against MEK1 (top panel) or MEK2 (the second panel). ERK activation was detected by antibodies against phosphorylated ERK (the third panel). Total ERK expression was detected by ERK antibody as a control (the fourth panel). Antibody against GAPDH was used as a loading control (bottom panel).
    Figure Legend Snippet: Necessity of MEK1 and MEK2 signaling pathways for ERK activation in SK-MEL-28 cells. SK-MEL-28 cells were transfected with nothing (N), lipid only (L), non-silencing control siRNA (NS), MEK-specific siRNA (MEK1 siRNA 7, 8, 11, and MEK2 siRNA 9, and 14), pools of MEK-specific siRNA (MEK1 siRNA 7+8+11 and MEK2 siRNA 9+14) or combinations of pools of MEK1- and MEK2-specific siRNA (P1, MEK1 siRNA 8+11 plus MEK2 siRNA 9+14; P2, MEK1 siRNA 7+8+11 plus MEK2 siRNA 9+14) as described in Material and Methods . After transfection, cells were trypsinized and split into separate dishes for cell lysate collection and for cell cycle analysis (results shown in Table 1 ). Seventy-two hours later, whole cell lysates were harvested and immunoblotted. The efficiency of siRNA-mediated MEK knock down was examined by immunoblotting with antibodies against MEK1 (top panel) or MEK2 (the second panel). ERK activation was detected by antibodies against phosphorylated ERK (the third panel). Total ERK expression was detected by ERK antibody as a control (the fourth panel). Antibody against GAPDH was used as a loading control (bottom panel).

    Techniques Used: Activation Assay, Transfection, Cell Cycle Assay, Expressing

    Individual MEK signaling in LeTx-treated SK-MEL-28 cells. SK-MEL-28 parental cells and cells stably expressing V5-MEK or V5-MEKcr were treated with LeTx (1 µg/ml PA plus 0, 1, 10, or 100 ng/ml LF) for 24 h. Total cell lysates were then harvested and immunoblotted with antibodies against the V5 epitope to confirm the cleavage resistance of V5-MEKcr (top panels). Antibodies against the carboxyl terminus of MEK1 (second panel) and the carboxyl terminus of MEK2 (third panel) were used to demonstrate individual MEK expression in LeTx-treated cells. Antibodies against phospho-ERK1/2 (fourth panel) and total ERK1/2 (fifth panel) were used to examine ERK activation, and an antibody against β-actin and β-tubulin were used as a loading control (bottom panels).
    Figure Legend Snippet: Individual MEK signaling in LeTx-treated SK-MEL-28 cells. SK-MEL-28 parental cells and cells stably expressing V5-MEK or V5-MEKcr were treated with LeTx (1 µg/ml PA plus 0, 1, 10, or 100 ng/ml LF) for 24 h. Total cell lysates were then harvested and immunoblotted with antibodies against the V5 epitope to confirm the cleavage resistance of V5-MEKcr (top panels). Antibodies against the carboxyl terminus of MEK1 (second panel) and the carboxyl terminus of MEK2 (third panel) were used to demonstrate individual MEK expression in LeTx-treated cells. Antibodies against phospho-ERK1/2 (fourth panel) and total ERK1/2 (fifth panel) were used to examine ERK activation, and an antibody against β-actin and β-tubulin were used as a loading control (bottom panels).

    Techniques Used: Stable Transfection, Expressing, Activation Assay

    31) Product Images from "Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling"

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Journal: Molecules

    doi: 10.3390/molecules21030301

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    Figure Legend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    32) Product Images from "Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation"

    Article Title: Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation

    Journal: Scientific Reports

    doi: 10.1038/srep17645

    Effect of fisetin or luteolin on signaling pathway proteins. Both fisetin and luteolin reduced the phosphorylation of CREB ( a ), p38 MAPK ( b ), ERK1/2 ( c ), and JNK ( d ), but not that of MEK1/2 ( e ) or the DNA-binding activity of NF-κB subunit p65 ( f ) when compared to cells treated with HNE and DMSO. Results are shown as scatterplots with median and are combined from 3-8 independent experiments with 4 parallel determinations per group/experiment. *denotes p
    Figure Legend Snippet: Effect of fisetin or luteolin on signaling pathway proteins. Both fisetin and luteolin reduced the phosphorylation of CREB ( a ), p38 MAPK ( b ), ERK1/2 ( c ), and JNK ( d ), but not that of MEK1/2 ( e ) or the DNA-binding activity of NF-κB subunit p65 ( f ) when compared to cells treated with HNE and DMSO. Results are shown as scatterplots with median and are combined from 3-8 independent experiments with 4 parallel determinations per group/experiment. *denotes p

    Techniques Used: Binding Assay, Activity Assay

    Effects of specific MAPK inhibitors on cell survival and cytokine release from HNE-treated serum-starved ARPE-19 cells. The JNK inhibitor SP600125 (SP), the MEK1/2 inhibitor PD98059 (PD) and the p38 MAPK inhibitor SB203580 (SB) had no protective effect on the cell viability (( a) : MTT assay and ( b ) LDH assay) of serum-starved ARPE-19 cells stimulated with HNE. Release of the cytokine IL-6 ( c ) was decreased only by PD, whereas IL-8 ( d ) levels were reduced by both PD and SP but increased by SB when compared to cells exposed to HNE and the solvent DMSO. Cell viability was normalized to untreated controls while the cytokine levels were compared to HNE + DMSO-exposed positive controls. Addition of HNE + DMSO reduced the release of IL-6 and IL-8 compared to untreated control ( Suppl. Fig. 4 ). Results are shown as scatterplots with median and are combined from 3 independent experiments with 3–4 parallel determinations per group/experiment. *denotes p
    Figure Legend Snippet: Effects of specific MAPK inhibitors on cell survival and cytokine release from HNE-treated serum-starved ARPE-19 cells. The JNK inhibitor SP600125 (SP), the MEK1/2 inhibitor PD98059 (PD) and the p38 MAPK inhibitor SB203580 (SB) had no protective effect on the cell viability (( a) : MTT assay and ( b ) LDH assay) of serum-starved ARPE-19 cells stimulated with HNE. Release of the cytokine IL-6 ( c ) was decreased only by PD, whereas IL-8 ( d ) levels were reduced by both PD and SP but increased by SB when compared to cells exposed to HNE and the solvent DMSO. Cell viability was normalized to untreated controls while the cytokine levels were compared to HNE + DMSO-exposed positive controls. Addition of HNE + DMSO reduced the release of IL-6 and IL-8 compared to untreated control ( Suppl. Fig. 4 ). Results are shown as scatterplots with median and are combined from 3 independent experiments with 3–4 parallel determinations per group/experiment. *denotes p

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay

    33) Product Images from "Blueberry Opposes ?-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase"

    Article Title: Blueberry Opposes ?-Amyloid Peptide-Induced Microglial Activation Via Inhibition of p44/42 Mitogen-Activation Protein Kinase

    Journal: Rejuvenation Research

    doi: 10.1089/rej.2008.0757

    BB suppresses microglial activation and enhances microglial phagocytosis of Aβ 1–42 peptide through a p44/42 MAPK-dependent pathway. Microglial treatment conditions are indicated and are further described in the Materials and Methods section.
    Figure Legend Snippet: BB suppresses microglial activation and enhances microglial phagocytosis of Aβ 1–42 peptide through a p44/42 MAPK-dependent pathway. Microglial treatment conditions are indicated and are further described in the Materials and Methods section.

    Techniques Used: Activation Assay

    34) Product Images from "p53 suppresses carcinoma progression by inhibiting mTOR pathway activation"

    Article Title: p53 suppresses carcinoma progression by inhibiting mTOR pathway activation

    Journal: Oncogene

    doi: 10.1038/onc.2013.589

    p53 loss results in mTOR pathway activation in Rb deficient MTC Activated, phosphorylated forms of S6 kinase (p-S6K), AKT (p-AKT) and MEK (p-MEK1/2) as well as corresponding total protein levels (S6K, AKT and MEK-1) were compared between Rb and Rb/p53 ablated thyroid tumors by Western blot analysis. (a) p-S6K was significantly increased in Rb/p53 as compared to Rb ablated tumors despite similar total S6K levels. (b) p-AKT and total AKT levels were similar in Rb and Rb/p53 ablated tumors. (c) p-MEK was significantly decreased in Rb/p53 as compared to Rb ablated tumors despite similar total MEK levels. Quantification is represented as relative densitometric values of phosphorylated:total protein ratios. (d) p53 loss in Rb deficient MTC results in decreased expression of mTOR pathway inhibitors. Schematic diagram illustrating previously identified p53 target genes (blue outlined boxes) known to repress mTOR signaling. Activation is indicated in blue and suppression in red. Quantitative RT-PCR showed significantly reduced expression of Sesn2, Tsc2, Plk2, Igfbp3 and Pten (blue shaded boxes), but not Ddit4 and Prkab1 (unshaded boxes), in Rb/p53 as compared to Rb ablated MTC. Data are represented as mean ± SD. *p
    Figure Legend Snippet: p53 loss results in mTOR pathway activation in Rb deficient MTC Activated, phosphorylated forms of S6 kinase (p-S6K), AKT (p-AKT) and MEK (p-MEK1/2) as well as corresponding total protein levels (S6K, AKT and MEK-1) were compared between Rb and Rb/p53 ablated thyroid tumors by Western blot analysis. (a) p-S6K was significantly increased in Rb/p53 as compared to Rb ablated tumors despite similar total S6K levels. (b) p-AKT and total AKT levels were similar in Rb and Rb/p53 ablated tumors. (c) p-MEK was significantly decreased in Rb/p53 as compared to Rb ablated tumors despite similar total MEK levels. Quantification is represented as relative densitometric values of phosphorylated:total protein ratios. (d) p53 loss in Rb deficient MTC results in decreased expression of mTOR pathway inhibitors. Schematic diagram illustrating previously identified p53 target genes (blue outlined boxes) known to repress mTOR signaling. Activation is indicated in blue and suppression in red. Quantitative RT-PCR showed significantly reduced expression of Sesn2, Tsc2, Plk2, Igfbp3 and Pten (blue shaded boxes), but not Ddit4 and Prkab1 (unshaded boxes), in Rb/p53 as compared to Rb ablated MTC. Data are represented as mean ± SD. *p

    Techniques Used: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

    35) Product Images from "β-amyloid cytotoxicity is prevented by natural achillolide A"

    Article Title: β-amyloid cytotoxicity is prevented by natural achillolide A

    Journal: Journal of Natural Medicines

    doi: 10.1007/s11418-018-1191-0

    Achillolide A attenuates the phosphorylation of p44/42 MAPK and SAPK/JNK induced by Aβ in N2a cells. Cells were either untreated or treated with Aβ only (25 μM) or Aβ + achillolide A for 30 min (p44/42 MAPK) or 40 min (SAPK/JNK). The levels of phosphorylated and total SAPK/JNK ( a ) and p44/42 MAPK ( b ) in cell extracts were determined by corresponding ELISA kits. The levels of the phosphorylated proteins were normalized to the levels of the total amount of the related proteins, and are presented as the mean ± SEM of two experiments ( n = 4) for SAPK/JNK, and three experiments ( n = 6) for p44/42 MAPK. The levels of the phosphorylated proteins were significantly lower in cells treated with both Aβ + achillolide A compared to cells treated with Aβ only. **p
    Figure Legend Snippet: Achillolide A attenuates the phosphorylation of p44/42 MAPK and SAPK/JNK induced by Aβ in N2a cells. Cells were either untreated or treated with Aβ only (25 μM) or Aβ + achillolide A for 30 min (p44/42 MAPK) or 40 min (SAPK/JNK). The levels of phosphorylated and total SAPK/JNK ( a ) and p44/42 MAPK ( b ) in cell extracts were determined by corresponding ELISA kits. The levels of the phosphorylated proteins were normalized to the levels of the total amount of the related proteins, and are presented as the mean ± SEM of two experiments ( n = 4) for SAPK/JNK, and three experiments ( n = 6) for p44/42 MAPK. The levels of the phosphorylated proteins were significantly lower in cells treated with both Aβ + achillolide A compared to cells treated with Aβ only. **p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    36) Product Images from "Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line"

    Article Title: Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)-retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2008.05409.x

    Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.
    Figure Legend Snippet: Schematic representation of the postulated MAP kinase signaling pathways involved in 4HPR-induced neuronal differentiation of ARPE-19 cells 4HPR mediates the neuronal differentiation of ARPE-19 cells by activating both c-Raf and MEK1/2, while the activation of its downstream targets such as SAPK/JNK and MAPK/ERK1/2 was regulated by MEK1/2. The activation of ERK1/2 appears to activate its downstream targets such as p90RSK and SAPK/JNK, and resulted in the phosphorylation of c-Fos and c-Jun, respectively. The activated form of c-Fos and c-Jun mediates the transactivation of AP-1, one of the effectors of differentiation, perhaps through an AP-1 response element present in the promoter of the neuronal marker calretinin. The question mark (?) indicates that the involvement of retinoid receptors in this process is not yet known.

    Techniques Used: Activation Assay, Marker

    MEK1/2 pathway mediates 4HPR-induced neuronal differentiation of ARPE-19 cells Cultured ARPE-19 cells were pretreated with 1 μM U0126 for 1 h followed by incubation with 1μM 4HPR for additional 72 h. Cell lysates were prepared, and then analyzed by Western blotting using non-phospho or phospho-specific antibodies of MAPK/ERK pathway as described under Materials and Methods. Panel A, 4HPR-induced phosphorylation of c-Raf and MEK1/2 were not blocked by U0126. Panel B , the inhibition of 4HPR-induced phosphorylation of p90RSK and c-Fos by U0126. Panel C, the inhibition of 4HPR-induced phosphorylation of SAPK/JNK and c-Jun by U0126.
    Figure Legend Snippet: MEK1/2 pathway mediates 4HPR-induced neuronal differentiation of ARPE-19 cells Cultured ARPE-19 cells were pretreated with 1 μM U0126 for 1 h followed by incubation with 1μM 4HPR for additional 72 h. Cell lysates were prepared, and then analyzed by Western blotting using non-phospho or phospho-specific antibodies of MAPK/ERK pathway as described under Materials and Methods. Panel A, 4HPR-induced phosphorylation of c-Raf and MEK1/2 were not blocked by U0126. Panel B , the inhibition of 4HPR-induced phosphorylation of p90RSK and c-Fos by U0126. Panel C, the inhibition of 4HPR-induced phosphorylation of SAPK/JNK and c-Jun by U0126.

    Techniques Used: Cell Culture, Incubation, Western Blot, Inhibition

    37) Product Images from "Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling"

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Journal: Molecules

    doi: 10.3390/molecules21030301

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    Figure Legend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    38) Product Images from "Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway"

    Article Title: Ginsenoside Rg1 activates ligand-independent estrogenic effects via rapid estrogen receptor signaling pathway

    Journal: Journal of Ginseng Research

    doi: 10.1016/j.jgr.2018.03.004

    Possible mechanisms of actions of Rg1 in ER positive cells . Rg1 (1) activates the recruitment of ERα to plasma membrane via the formation of signalsome containing caveolin, Shc, MNAR, IGFR, and c-Src and induces an increase in intracellular Ca mobilization; (2) activates GPER–EGFR cross talk and induces an increase in intracellular cAMP; and (3) leads to an increase in MEK1/2 phosphorylation and ERα phosphorylation at Ser118, followed by an induction of ERE-dependent transcription and finally the exertion of estrogenic effects.
    Figure Legend Snippet: Possible mechanisms of actions of Rg1 in ER positive cells . Rg1 (1) activates the recruitment of ERα to plasma membrane via the formation of signalsome containing caveolin, Shc, MNAR, IGFR, and c-Src and induces an increase in intracellular Ca mobilization; (2) activates GPER–EGFR cross talk and induces an increase in intracellular cAMP; and (3) leads to an increase in MEK1/2 phosphorylation and ERα phosphorylation at Ser118, followed by an induction of ERE-dependent transcription and finally the exertion of estrogenic effects.

    Techniques Used:

    39) Product Images from "Targeting Pin1 by All-Trans Retinoic Acid (ATRA) Overcomes Tamoxifen Resistance in Breast Cancer via Multifactorial Mechanisms"

    Article Title: Targeting Pin1 by All-Trans Retinoic Acid (ATRA) Overcomes Tamoxifen Resistance in Breast Cancer via Multifactorial Mechanisms

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2019.00322

    ATRA suppresses nuclear expression and transactivation of ERα. (A,B) ATRA decreases the level of nuclear ERα in MCF-7 and MCF-7R. Cells were treated with 10 μM ATRA for 72 h before immunofluorescence staining. Scale bars, 50 μm. (C,D) ATRA decreases the level of nuclear ERα in T47D and T47DR. Cells were treated with10 μM ATRA for 72 h before immunofluorescence staining. Scale bars, 50 μm. (E–H) ATRA suppresses the transcription of ERα target genes GREB1, PGR, and c-Myc. Cells were treated with ATRA (2.5, 5.0, and10 μM) for 48 h. Expression of ERα downstream genes were detected by qRT-PCR, and normalized to β-ACTIN expression in DMSO treated cells. Error bars denote the SD of three biological replicates, ∗ P
    Figure Legend Snippet: ATRA suppresses nuclear expression and transactivation of ERα. (A,B) ATRA decreases the level of nuclear ERα in MCF-7 and MCF-7R. Cells were treated with 10 μM ATRA for 72 h before immunofluorescence staining. Scale bars, 50 μm. (C,D) ATRA decreases the level of nuclear ERα in T47D and T47DR. Cells were treated with10 μM ATRA for 72 h before immunofluorescence staining. Scale bars, 50 μm. (E–H) ATRA suppresses the transcription of ERα target genes GREB1, PGR, and c-Myc. Cells were treated with ATRA (2.5, 5.0, and10 μM) for 48 h. Expression of ERα downstream genes were detected by qRT-PCR, and normalized to β-ACTIN expression in DMSO treated cells. Error bars denote the SD of three biological replicates, ∗ P

    Techniques Used: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR

    40) Product Images from "Sphingosine Kinase-1 Is Required for Toll Mediated ?-Defensin 2 Induction in Human Oral Keratinocytes"

    Article Title: Sphingosine Kinase-1 Is Required for Toll Mediated ?-Defensin 2 Induction in Human Oral Keratinocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011512

    Inhibition of GSK3 augments HBD-2 induction. Keratinocytes were pretreated with PI3K inhibitor (LY294002–10 µM and wortmannin –0.5 µM) or Akt inhibitor (10 µM) or MEK 1/2, U0126 (25 uM) or GSK3 inhibitor (SB216763-12 µM) 2 h before challenging with FSL-1(1 µg/ml) for 24 h. The supernatant was subjected to human HBD-2 ELISA. PI3K, Akt and MEK inhibitors abrogated HBD-2 induction whereas GSK3 inhibitor augmented HBD-2 induction in HGECs ( A ). The cells were pretreated with LY294002 (10 µM) for 2 h and challenged with FSL-1 (1 µg/ml) for 0, 30, 60, 90, 120 and 240 min. Total protein was subjected to immunoblot against phospho- ser225 Sphk-1 antibody with β-actin as loading control. PI3K inhibitor downregulated the phosphorylation level of Sphk-1 at Ser225 demonstrating the pivotal role of PI3K in Sphk-1 activation ( B ). The cells were transient transfected of siRNA against GSK-3β and challenged with FSL-1 (1 µg/ml) for 24 h and supernatants were subjected to human HBD-2 ELISA. siGSK3-β up regulated HBD-2 induction after challenging with FSL-1. This upregulation of HBD-2 was significantly higher to FSL-1 challenge alone ( C ). The cells were either pretreated with U0126 (25 uM) and/or SB216763 (12 µM) prior to challenging with FSL-1 (1 µg/ml) for 24 h and supernatant was subjected to HBD-2 ELISA. GSK3 inhibitor augmented the HBD-2 induction whereas the cells with GSK3 +MEK 1/2 inhibitor ablated HBD-2 induction ( D ). Phospho-Glycogen synthase (Ser641) levels were assessed by incubating SB216763 (12 µM) for 2 h prior to challenge with FSL-1 for 0, 30 and 60 min. Total protein was subjected to immunoblot against Phospho-Glycogen synthase (Ser641) antibody with β-actin as loading control. The phosphorylation of Glycogen synthase at Ser641 was down regulated in the presence of SB216763 ( E ). Time course experiment was performed either in the presence or absence of SB 216763 inhibitor (GSK3) (12 µM). The cells were challenged with FSL-1 (1 µg/ml) and the total protein was collected at 15, 30 and 60 min and subjected to immunoblot against phospho- ser225 Sphk-1 antibody and p44/42 MAPK (Erk1/2) antibody with β-actin as loading control. Inhibition of GSK3 by SB 216763 increased the Sphk-1 phospho-ser225 at 60 min, ERK 1/2 phosphorylation increased at 30 min of agonist challenge ( F ). The phosphorylation of p44/42 MAPK (Erk1/2) was unaltered upon Sphk-1 inhibitor after 60 min demonstrating Sphk-1 downstream of Erk 1/2 ( G ). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p
    Figure Legend Snippet: Inhibition of GSK3 augments HBD-2 induction. Keratinocytes were pretreated with PI3K inhibitor (LY294002–10 µM and wortmannin –0.5 µM) or Akt inhibitor (10 µM) or MEK 1/2, U0126 (25 uM) or GSK3 inhibitor (SB216763-12 µM) 2 h before challenging with FSL-1(1 µg/ml) for 24 h. The supernatant was subjected to human HBD-2 ELISA. PI3K, Akt and MEK inhibitors abrogated HBD-2 induction whereas GSK3 inhibitor augmented HBD-2 induction in HGECs ( A ). The cells were pretreated with LY294002 (10 µM) for 2 h and challenged with FSL-1 (1 µg/ml) for 0, 30, 60, 90, 120 and 240 min. Total protein was subjected to immunoblot against phospho- ser225 Sphk-1 antibody with β-actin as loading control. PI3K inhibitor downregulated the phosphorylation level of Sphk-1 at Ser225 demonstrating the pivotal role of PI3K in Sphk-1 activation ( B ). The cells were transient transfected of siRNA against GSK-3β and challenged with FSL-1 (1 µg/ml) for 24 h and supernatants were subjected to human HBD-2 ELISA. siGSK3-β up regulated HBD-2 induction after challenging with FSL-1. This upregulation of HBD-2 was significantly higher to FSL-1 challenge alone ( C ). The cells were either pretreated with U0126 (25 uM) and/or SB216763 (12 µM) prior to challenging with FSL-1 (1 µg/ml) for 24 h and supernatant was subjected to HBD-2 ELISA. GSK3 inhibitor augmented the HBD-2 induction whereas the cells with GSK3 +MEK 1/2 inhibitor ablated HBD-2 induction ( D ). Phospho-Glycogen synthase (Ser641) levels were assessed by incubating SB216763 (12 µM) for 2 h prior to challenge with FSL-1 for 0, 30 and 60 min. Total protein was subjected to immunoblot against Phospho-Glycogen synthase (Ser641) antibody with β-actin as loading control. The phosphorylation of Glycogen synthase at Ser641 was down regulated in the presence of SB216763 ( E ). Time course experiment was performed either in the presence or absence of SB 216763 inhibitor (GSK3) (12 µM). The cells were challenged with FSL-1 (1 µg/ml) and the total protein was collected at 15, 30 and 60 min and subjected to immunoblot against phospho- ser225 Sphk-1 antibody and p44/42 MAPK (Erk1/2) antibody with β-actin as loading control. Inhibition of GSK3 by SB 216763 increased the Sphk-1 phospho-ser225 at 60 min, ERK 1/2 phosphorylation increased at 30 min of agonist challenge ( F ). The phosphorylation of p44/42 MAPK (Erk1/2) was unaltered upon Sphk-1 inhibitor after 60 min demonstrating Sphk-1 downstream of Erk 1/2 ( G ). Control cells received DMSO unless otherwise stated. Results are mean ± SEM and are representative of three independent experiments. Statistical comparisons are shown by horizontal bars with asterisks above them (* indicates p

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Activation Assay, Transfection

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    Article Snippet: .. For co-localization studies, after primary antibody incubation, sections were incubated with corresponding Alexa Fluor® secondary antibodies (1:2000, 1 hour, ThermoFisher) and DRAQ5® (1:2000, 10 minutes, Cell Signaling, MA, USA) nuclear stain. .. Fluorescence images were captured with a Leica TCS SP5 multiphoton laser scanning confocal microscope.

    Incubation:

    Article Title: Reduced RhoA expression enhances breast cancer metastasis with a concomitant increase in CCR5 and CXCR4 chemokines signaling
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    Article Snippet: .. The primary antibody for STAT3 (1:2000, Cell Signaling) or pSTAT3 (1:2000, Cell Signaling) was diluted in blocking buffer, applied and incubated overnight at 4 °C. .. After the washing with TBST, membrane was incubated with secondary antibody (Goat anti-rabbit HRP conjugated IgG, 1:5000, Jackson, USA) at room temperature for one hour followed by 2 min incubation ECL Western Blotting Substrate.

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    Article Snippet: .. After blocking in phosphate buffered saline (PBS) containing 0.05% Tween-20 and 5% non-fat milk powder, the membranes were incubated with the following primary antibodies: RNF8 (Santa Cruz Biotech, 1:500), EMT kit (Cell Signaling Technology, 1:2000), beta-actin (Santa Cruz Biotech, 1:4000), Secondary antibodies were Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (ZB-2305, ZSGB-Bio, 1:4000) or anti-Rabbit IgG(Fc) (ZB-2301, ZSGB-Bio, 1:4000). .. Subsequent visualization was detected by Digit imaging system (Thermo, Japan), the gray level of the bands was quantitated by ImageJ software.

    Immunofluorescence:

    Article Title: Dysfunction of the ubiquitin ligase E3A Ube3A/E6-AP contributes to synaptic pathology in Alzheimer’s disease
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    Blocking Assay:

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    Western Blot:

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    Cell Signaling Technology Inc phospho mek1 2
    MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active <t>MEK1/2</t> (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.
    Phospho Mek1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ARL11 depletion in macrophages results in defective killing of intracellular Salmonella . a , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total <t>ERK1/2,</t> p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control and Arl11 -silenced RAW264.7 cells infected with Salmonella for different time periods, as indicated. e–g , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, supernatants from the cultures were collected, the concentration of IL-6 ( e ) and TNFα ( f ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( g ). h and i , control, Arl11 -silenced, and Arl11 rescue RAW264.7 cells were infected with Salmonella , and the -fold change in recoverable cfu was calculated (20 h/2 h p.i.) by a gentamicin protection assay. By using confocal microscopy, the intracellular bacteria were counted in ∼100 cells/experiment. These numbers were grouped according to the key and expressed as a percentage of the total infected cell population ( i ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p
    Phospho Erk1 2 Pathway Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and <t>MEK1</t> in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by <t>ELISA.</t> Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
    Pathscan Total Mek1 Sandwich Elisa Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.

    Journal: Toxicology and applied pharmacology

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    doi: 10.1016/j.taap.2008.08.007

    Figure Lengend Snippet: MKP-3 knockdown via siRNA increases ERK1/2 phosphorylation in H- ras MCF10A and DLD-1 cells. (A) Whole cell lysates were prepared from serum starved MCF10A and H- ras MCF10A cells. MKP-3 ( top panel ) was detected by immunoblot analysis. The blot was stripped and reprobed for β-tubulin ( bottom panel ) as a loading control. Whole cell lysates were prepared from (B) H- ras MCF10A cells or (C) DLD-1 cells that were either not transfected (lane 1), were transfected with duplex siRNA targeted against MKP-3 (lane 2), or were transfected with duplex scrambled siRNA (lane 3). The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. As a positive control for phospho-MEK1/2, nontransfected cells were incubated for 120 minutes with (B) 1.6 nM TPA (lane 4), or incubated for 30 minutes with (C) 3 nM EGF (lane 4). ). The graphs represent average pERK1/2 densitometry relative to non-transfected controls for (B) 10 independent experiments and (C) 6 independent experiments; error bars represent SEM. Filled bars (nt), not transfected. Open bars (M), transfected with duplex siRNA targeted against MKP-3. Hatched bars (S), transfected with duplex scrambled siRNA. Treatment of H- ras MCF10A and DLD-1 cells with MKP3 siRNA resulted in an increase in pERK1/2 levels relative to nontransfected control or treatment with scrambled siRNA (H- ras MCF10A: p = 0.006, n=30, 1-way ANOVA and Tukey’s HSD, α=0.05; DLD-1: p= 0.0008, n=18, 1-way ANOVA and Tukey’s HSD, α=0.05). We did not detect a difference between non transfected cells or cells transfected with scrambled siRNA.

    Article Snippet: After blocking in a TBST/5% milk solution, immunoblots were incubated overnight at 4°C using the following primary antibodies and dilutions: Phospho-p44/42 MAPK (Thr-202/Tyr-204) (E10) (mouse monoclonal) (1:2000), and phospho-MEK1/2 (Ser-217/221) (rabbit polyclonal) (1:2000) from Cell Signaling (Beverly, MA), and ERK2 (C-14) (rabbit polyclonal) (1:2000), MEK1 (12-B) (rabbit polyclonal) (1:500), MKP-3 (C-20) (goat polyclonal) (1:1000) and β-tubulin (H-235) (rabbit polyclonal) (1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Transfection, Positive Control, Incubation

    TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.

    Journal: Toxicology and applied pharmacology

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    doi: 10.1016/j.taap.2008.08.007

    Figure Lengend Snippet: TPA and EGF stimulate the loss and recovery of MKP-3 protein. Whole cell lysates were prepared from (A) H- ras MCF10A cells that were incubated for the indicated times with 1.6 nM TPA; (B) H- ras MCF10A cells; or (C) DLD-1 cells that were incubated for the indicated times with 3 nM EGF. The following proteins were detected by immunoblot analysis: dually phosphorylated, active MEK1/2 (pMEK1/2); total MEK1; dually phosphorylated, active ERK1/2 (pERK1/2); total ERK2; or MKP-3. The MKP-3 blot was stripped and reprobed for β-tubulin as a loading control. The graphs represent average densitometry of pMEK1/2, pERK1/2, and MKP-3 for 3 independent experiments; error bars represent SEM. Filled bars, pMEK1/2. Open bars, pERK1/2. Hatched bars, MKP-3.

    Article Snippet: After blocking in a TBST/5% milk solution, immunoblots were incubated overnight at 4°C using the following primary antibodies and dilutions: Phospho-p44/42 MAPK (Thr-202/Tyr-204) (E10) (mouse monoclonal) (1:2000), and phospho-MEK1/2 (Ser-217/221) (rabbit polyclonal) (1:2000) from Cell Signaling (Beverly, MA), and ERK2 (C-14) (rabbit polyclonal) (1:2000), MEK1 (12-B) (rabbit polyclonal) (1:500), MKP-3 (C-20) (goat polyclonal) (1:1000) and β-tubulin (H-235) (rabbit polyclonal) (1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Incubation

    Reciprocal regulation of ERK1/2 and MKP-3. Phosphorylation and activation of ERK1/2 typically occurs through stimulation of the Ras/Raf/MEK1/2 protein kinase cascade. The GTPase Ras activates the protein kinase Raf, which phosphorylates and activates the protein kinases MEK1/2. MEK1/2 is highly specific for phosphorylating and activating ERK1/2. The magnitude and duration of ERK1/2 activity is determined by the balance between the activity of MEK1/2, the kinases that phosphorylate and activate ERK1/2, and the activity of phosphatases, such as MKP-3, which dephosphorylate and inactivate ERK1/2. The levels of MKP-3 protein available to inhibit ERK1/2 are regulated, at least in part, by ERK1/2. ERK1/2 can stimulate an increase in the expression of MKP-3 RNA. This results in an increase in MKP-3 protein, which can then feedback and inhibit ERK1/2 activity. ERK1/2 can also stimulate a decrease in MKP-3 protein stability, however, which can cause a loss of MKP-3 protein. Thus, the levels of MKP-3 protein are determined, at least in part, by the balance between the stimulation of new MKP-3 gene expression and the stability of the MKP-3 protein.

    Journal: Toxicology and applied pharmacology

    Article Title: Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3

    doi: 10.1016/j.taap.2008.08.007

    Figure Lengend Snippet: Reciprocal regulation of ERK1/2 and MKP-3. Phosphorylation and activation of ERK1/2 typically occurs through stimulation of the Ras/Raf/MEK1/2 protein kinase cascade. The GTPase Ras activates the protein kinase Raf, which phosphorylates and activates the protein kinases MEK1/2. MEK1/2 is highly specific for phosphorylating and activating ERK1/2. The magnitude and duration of ERK1/2 activity is determined by the balance between the activity of MEK1/2, the kinases that phosphorylate and activate ERK1/2, and the activity of phosphatases, such as MKP-3, which dephosphorylate and inactivate ERK1/2. The levels of MKP-3 protein available to inhibit ERK1/2 are regulated, at least in part, by ERK1/2. ERK1/2 can stimulate an increase in the expression of MKP-3 RNA. This results in an increase in MKP-3 protein, which can then feedback and inhibit ERK1/2 activity. ERK1/2 can also stimulate a decrease in MKP-3 protein stability, however, which can cause a loss of MKP-3 protein. Thus, the levels of MKP-3 protein are determined, at least in part, by the balance between the stimulation of new MKP-3 gene expression and the stability of the MKP-3 protein.

    Article Snippet: After blocking in a TBST/5% milk solution, immunoblots were incubated overnight at 4°C using the following primary antibodies and dilutions: Phospho-p44/42 MAPK (Thr-202/Tyr-204) (E10) (mouse monoclonal) (1:2000), and phospho-MEK1/2 (Ser-217/221) (rabbit polyclonal) (1:2000) from Cell Signaling (Beverly, MA), and ERK2 (C-14) (rabbit polyclonal) (1:2000), MEK1 (12-B) (rabbit polyclonal) (1:500), MKP-3 (C-20) (goat polyclonal) (1:1000) and β-tubulin (H-235) (rabbit polyclonal) (1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Activity Assay, Expressing

    ARL11 depletion in macrophages results in defective killing of intracellular Salmonella . a , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control and Arl11 -silenced RAW264.7 cells infected with Salmonella for different time periods, as indicated. e–g , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, supernatants from the cultures were collected, the concentration of IL-6 ( e ) and TNFα ( f ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( g ). h and i , control, Arl11 -silenced, and Arl11 rescue RAW264.7 cells were infected with Salmonella , and the -fold change in recoverable cfu was calculated (20 h/2 h p.i.) by a gentamicin protection assay. By using confocal microscopy, the intracellular bacteria were counted in ∼100 cells/experiment. These numbers were grouped according to the key and expressed as a percentage of the total infected cell population ( i ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 depletion in macrophages results in defective killing of intracellular Salmonella . a , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control and Arl11 -silenced RAW264.7 cells infected with Salmonella for different time periods, as indicated. e–g , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, supernatants from the cultures were collected, the concentration of IL-6 ( e ) and TNFα ( f ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( g ). h and i , control, Arl11 -silenced, and Arl11 rescue RAW264.7 cells were infected with Salmonella , and the -fold change in recoverable cfu was calculated (20 h/2 h p.i.) by a gentamicin protection assay. By using confocal microscopy, the intracellular bacteria were counted in ∼100 cells/experiment. These numbers were grouped according to the key and expressed as a percentage of the total infected cell population ( i ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Article Snippet: The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti- Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

    ARL11 overexpression in macrophages was sufficient for ERK1/2 phosphorylation and impaired further stimulation of macrophages upon LPS stimulation. a , RAW264.7 cells were transfected with plasmid encoding ARL11-TAP or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. The overexpression of ARL11-TAP was confirmed by probing with anti-TAP antibody. *, nonspecific protein band. b , representative phase-contrast micrographs of control and ARL11-TAP–overexpressing RAW264.7 cells. Overexpression of ARL11 leads to multiple-pseudopodia formation, a hallmark of activated macrophages. Bar , 10 μm. c and d , HeLa ( c ) and A549 ( d ) cells were transfected with the indicated ARL-encoding plasmids or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. e and f , overexpression of ARL11 inhibited LPS-induced production of pro-inflammatory cytokines. RAW264.7 cells expressing ARL11-TAP or empty vector–transfected were stimulated with 1 μg/ml LPS for the indicated time periods. Supernatants from the cultures were collected, and the concentration of IL-6 ( e ) and TNFα ( f ) was evaluated by ELISA. Data shown represent mean ± S.D. ( error bars ) ( n = 3) (****, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 overexpression in macrophages was sufficient for ERK1/2 phosphorylation and impaired further stimulation of macrophages upon LPS stimulation. a , RAW264.7 cells were transfected with plasmid encoding ARL11-TAP or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. The overexpression of ARL11-TAP was confirmed by probing with anti-TAP antibody. *, nonspecific protein band. b , representative phase-contrast micrographs of control and ARL11-TAP–overexpressing RAW264.7 cells. Overexpression of ARL11 leads to multiple-pseudopodia formation, a hallmark of activated macrophages. Bar , 10 μm. c and d , HeLa ( c ) and A549 ( d ) cells were transfected with the indicated ARL-encoding plasmids or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. e and f , overexpression of ARL11 inhibited LPS-induced production of pro-inflammatory cytokines. RAW264.7 cells expressing ARL11-TAP or empty vector–transfected were stimulated with 1 μg/ml LPS for the indicated time periods. Supernatants from the cultures were collected, and the concentration of IL-6 ( e ) and TNFα ( f ) was evaluated by ELISA. Data shown represent mean ± S.D. ( error bars ) ( n = 3) (****, p

    Article Snippet: The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti- Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

    ARL11 depletion impairs ERK1/2 and p38 MAPK phosphorylation in LPS-stimulated macrophages. a , control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells were treated with 1 μg/ml LPS for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells treated with 1 μg/ml LPS for different time periods, as indicated. e , BMDMs were transfected with control or Arl11 siRNA. After 72 h of siRNA transfections, cells were stimulated with 100 ng/ml LPS for the indicated time periods, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. f–h , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( f ), phospho-p38 to total p38 ( g ), and phospho-JNK to total JNK ( h ) in control siRNA– and Arl11 siRNA–transfected BMDMs treated with 100 ng/ml LPS for different time periods as indicated. i , cell lysates of control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. To confirm the expression of human ARL11 -HA rescue plasmid, the lysates were probed with anti-HA antibody. j–l , control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS for the indicated time periods, supernatants from the cultures were collected, the concentration of IL-6 ( j ) and TNFα ( k ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( l ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 depletion impairs ERK1/2 and p38 MAPK phosphorylation in LPS-stimulated macrophages. a , control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells were treated with 1 μg/ml LPS for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells treated with 1 μg/ml LPS for different time periods, as indicated. e , BMDMs were transfected with control or Arl11 siRNA. After 72 h of siRNA transfections, cells were stimulated with 100 ng/ml LPS for the indicated time periods, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. f–h , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( f ), phospho-p38 to total p38 ( g ), and phospho-JNK to total JNK ( h ) in control siRNA– and Arl11 siRNA–transfected BMDMs treated with 100 ng/ml LPS for different time periods as indicated. i , cell lysates of control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. To confirm the expression of human ARL11 -HA rescue plasmid, the lysates were probed with anti-HA antibody. j–l , control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS for the indicated time periods, supernatants from the cultures were collected, the concentration of IL-6 ( j ) and TNFα ( k ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( l ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Article Snippet: The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti- Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: shRNA, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    ARL11 depletion in macrophages results in defective killing of intracellular Salmonella . a , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control and Arl11 -silenced RAW264.7 cells infected with Salmonella for different time periods, as indicated. e–g , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, supernatants from the cultures were collected, the concentration of IL-6 ( e ) and TNFα ( f ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( g ). h and i , control, Arl11 -silenced, and Arl11 rescue RAW264.7 cells were infected with Salmonella , and the -fold change in recoverable cfu was calculated (20 h/2 h p.i.) by a gentamicin protection assay. By using confocal microscopy, the intracellular bacteria were counted in ∼100 cells/experiment. These numbers were grouped according to the key and expressed as a percentage of the total infected cell population ( i ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 depletion in macrophages results in defective killing of intracellular Salmonella . a , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control and Arl11 -silenced RAW264.7 cells infected with Salmonella for different time periods, as indicated. e–g , control and Arl11 -silenced RAW264.7 cells were infected with Salmonella for different time periods, supernatants from the cultures were collected, the concentration of IL-6 ( e ) and TNFα ( f ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( g ). h and i , control, Arl11 -silenced, and Arl11 rescue RAW264.7 cells were infected with Salmonella , and the -fold change in recoverable cfu was calculated (20 h/2 h p.i.) by a gentamicin protection assay. By using confocal microscopy, the intracellular bacteria were counted in ∼100 cells/experiment. These numbers were grouped according to the key and expressed as a percentage of the total infected cell population ( i ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Article Snippet: Antibodies and chemicals The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti-Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

    ARL11 overexpression in macrophages was sufficient for ERK1/2 phosphorylation and impaired further stimulation of macrophages upon LPS stimulation. a , RAW264.7 cells were transfected with plasmid encoding ARL11-TAP or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. The overexpression of ARL11-TAP was confirmed by probing with anti-TAP antibody. *, nonspecific protein band. b , representative phase-contrast micrographs of control and ARL11-TAP–overexpressing RAW264.7 cells. Overexpression of ARL11 leads to multiple-pseudopodia formation, a hallmark of activated macrophages. Bar , 10 μm. c and d , HeLa ( c ) and A549 ( d ) cells were transfected with the indicated ARL-encoding plasmids or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. e and f , overexpression of ARL11 inhibited LPS-induced production of pro-inflammatory cytokines. RAW264.7 cells expressing ARL11-TAP or empty vector–transfected were stimulated with 1 μg/ml LPS for the indicated time periods. Supernatants from the cultures were collected, and the concentration of IL-6 ( e ) and TNFα ( f ) was evaluated by ELISA. Data shown represent mean ± S.D. ( error bars ) ( n = 3) (****, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 overexpression in macrophages was sufficient for ERK1/2 phosphorylation and impaired further stimulation of macrophages upon LPS stimulation. a , RAW264.7 cells were transfected with plasmid encoding ARL11-TAP or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. The overexpression of ARL11-TAP was confirmed by probing with anti-TAP antibody. *, nonspecific protein band. b , representative phase-contrast micrographs of control and ARL11-TAP–overexpressing RAW264.7 cells. Overexpression of ARL11 leads to multiple-pseudopodia formation, a hallmark of activated macrophages. Bar , 10 μm. c and d , HeLa ( c ) and A549 ( d ) cells were transfected with the indicated ARL-encoding plasmids or empty vector. After 16 h, cells were lysed, and lysates were prepared and blotted with the indicated antibodies. e and f , overexpression of ARL11 inhibited LPS-induced production of pro-inflammatory cytokines. RAW264.7 cells expressing ARL11-TAP or empty vector–transfected were stimulated with 1 μg/ml LPS for the indicated time periods. Supernatants from the cultures were collected, and the concentration of IL-6 ( e ) and TNFα ( f ) was evaluated by ELISA. Data shown represent mean ± S.D. ( error bars ) ( n = 3) (****, p

    Article Snippet: Antibodies and chemicals The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti-Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

    ARL11 depletion impairs ERK1/2 and p38 MAPK phosphorylation in LPS-stimulated macrophages. a , control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells were treated with 1 μg/ml LPS for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells treated with 1 μg/ml LPS for different time periods, as indicated. e , BMDMs were transfected with control or Arl11 siRNA. After 72 h of siRNA transfections, cells were stimulated with 100 ng/ml LPS for the indicated time periods, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. f–h , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( f ), phospho-p38 to total p38 ( g ), and phospho-JNK to total JNK ( h ) in control siRNA– and Arl11 siRNA–transfected BMDMs treated with 100 ng/ml LPS for different time periods as indicated. i , cell lysates of control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. To confirm the expression of human ARL11 -HA rescue plasmid, the lysates were probed with anti-HA antibody. j–l , control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS for the indicated time periods, supernatants from the cultures were collected, the concentration of IL-6 ( j ) and TNFα ( k ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( l ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

    doi: 10.1074/jbc.RA117.000727

    Figure Lengend Snippet: ARL11 depletion impairs ERK1/2 and p38 MAPK phosphorylation in LPS-stimulated macrophages. a , control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells were treated with 1 μg/ml LPS for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. b–d , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( b ), phospho-p38 to total p38 ( c ), and phospho-JNK to total JNK ( d ) in control shRNA– and Arl11 shRNA 1–transfected RAW264.7 cells treated with 1 μg/ml LPS for different time periods, as indicated. e , BMDMs were transfected with control or Arl11 siRNA. After 72 h of siRNA transfections, cells were stimulated with 100 ng/ml LPS for the indicated time periods, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. f–h , densitometric analysis was performed to determine the ratio of phospho-ERK to total ERK ( f ), phospho-p38 to total p38 ( g ), and phospho-JNK to total JNK ( h ) in control siRNA– and Arl11 siRNA–transfected BMDMs treated with 100 ng/ml LPS for different time periods as indicated. i , cell lysates of control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS, and the lysates were prepared and blotted with the indicated anti-phospho-antibodies. Total ERK1/2, p38, JNK1/2, and α-tubulin were probed as quantitative controls. To confirm the expression of human ARL11 -HA rescue plasmid, the lysates were probed with anti-HA antibody. j–l , control shRNA–, Arl11 shRNA–, and Arl11 shRNA (rescue)–RAW264.7 cells were treated with LPS for the indicated time periods, supernatants from the cultures were collected, the concentration of IL-6 ( j ) and TNFα ( k ) was measured by ELISA, and nitrite production (an indication of the presence of nitric oxide) was evaluated by the Griess reaction ( l ). Data shown represent mean ± S.D. ( error bars ) ( n = 3) ( n.s. , not significant; *, p

    Article Snippet: Antibodies and chemicals The following antibodies were used in this study: mouse anti-HA (MMS-101P; Covance), rabbit anti-HA (H6908; Sigma-Aldrich), mouse anti-Myc (sc-40; Santa Cruz Biotechnology, Inc.), rabbit anti-CBP (07-482; Millipore), rabbit anti-Salmonella O-antigen (225341; BD Biosciences), rabbit anti-α-tubulin (ab15246; Abcam), mouse anti-GAPDH (sc-166574; Santa Cruz Biotechnology, Inc.), rabbit anti-histone H3 (9715; Cell Signaling Technology), rabbit anti-iNOS (sc-8310; Santa Cruz Biotechnology), mouse anti-CHOP (2895; Cell Signaling Technology), mouse anti-HO-1 (ADI-OSA-110-F; Enzo Life Sciences), rabbit anti-ARL11 (sc-83982; Santa Cruz Biotechnology), PE-conjugated rat anti-TLR4 (CD284) (145403; BioLegend), PE-conjugated rat anti-IgG1 (400508; BioLegend), rabbit anti-phospho-MEK1/2 (9154; Cell Signaling Technology), rabbit anti-MEK1/2 (9122; Cell Signaling Technology), rabbit anti-phospho-MKK3/MKK6 (9231; Cell Signaling Technology), rabbit anti-MKK3 (8535; Cell Signaling Technology), rabbit anti-phospho-p90RSK (11989; Cell Signaling Technology), rabbit anti-p90RSK (9355; Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (3033; Cell Signaling Technology), rabbit anti-NF-κB p65 (8242; Cell Signaling Technology), mouse anti-IκBα (4814; Cell Signaling Technology), MAPK family antibody kit (9926; Cell Signaling Technology), phospho-ERK1/2 pathway kit (9911; Cell Signaling Technology), phospho-MAPK family antibody kit (9910; Cell Signaling Technology), and apoptosis antibody kit (9915; Cell Signaling Technology).

    Techniques: shRNA, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Journal: Molecules

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    doi: 10.3390/molecules21030301

    Figure Lengend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Article Snippet: To measure the amount of total and phosphorylated MEK1 in cell lysates of astrocytes, ELISA was performed according to the manufacturer’s protocol using the PathScan total MEK1 sandwich ELISA kit (Cell Signaling Technology) and the PathScan phosoho-MEK1 (Ser217/221) sandwich ELISA kit (Cell Signaling Technology), respectively.

    Techniques: Enzyme-linked Immunosorbent Assay