rac1  (Millipore)


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    Name:
    Anti RAC1 antibody
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    Catalog Number:
    gw22212f
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    Structured Review

    Millipore rac1
    Effect of GRB7 on the downstream effectors of integrin stimulation in HER2 overexpressed breast cancer cell lines: A. Integrin (α4β1/α5β1)-induced activation of <t>RAC1.</t> BT474 overexpressing HER2 and trastuzumab-resistant

    https://www.bioz.com/result/rac1/product/Millipore
    Average 96 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    rac1 - by Bioz Stars, 2020-09
    96/100 stars

    Images

    1) Product Images from "Dissecting GRB7-mediated signals for proliferation and migration in HER2 overexpressing breast tumor cells: GTP-ase rules"

    Article Title: Dissecting GRB7-mediated signals for proliferation and migration in HER2 overexpressing breast tumor cells: GTP-ase rules

    Journal: American Journal of Cancer Research

    doi:

    Effect of GRB7 on the downstream effectors of integrin stimulation in HER2 overexpressed breast cancer cell lines: A. Integrin (α4β1/α5β1)-induced activation of RAC1. BT474 overexpressing HER2 and trastuzumab-resistant
    Figure Legend Snippet: Effect of GRB7 on the downstream effectors of integrin stimulation in HER2 overexpressed breast cancer cell lines: A. Integrin (α4β1/α5β1)-induced activation of RAC1. BT474 overexpressing HER2 and trastuzumab-resistant

    Techniques Used: Activation Assay

    2) Product Images from "Zyxin is involved in regulation of mechanotransduction in arteriole smooth muscle cells"

    Article Title: Zyxin is involved in regulation of mechanotransduction in arteriole smooth muscle cells

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2012.00472

    Zyxin silencing increased FAK and TRIP6 expression, but decreased Rac1 expression in VSMC. (A) Western blots showed that FAK and TRIP6 expression were increased, but Rac1 was decreased in cells stably expressing zyxin-shRNA compared to cells expressing non-silencing control. Blots were quantified and normalized to β-actin and presented as percentage of the control. (B) Immunofluorescence microscopy showed increased presence of TRIP6 in the focal adhesions of VSMCs expressing zyxin-shRNA, but no significant change was observed in the focal adhesion presence of paxillin and vinculin. Control and Zyxin-silenced samples were run on the same immunoblot and were from the same film exposure. The results are representative of three independent experiments. Scale bars, 30 μm.
    Figure Legend Snippet: Zyxin silencing increased FAK and TRIP6 expression, but decreased Rac1 expression in VSMC. (A) Western blots showed that FAK and TRIP6 expression were increased, but Rac1 was decreased in cells stably expressing zyxin-shRNA compared to cells expressing non-silencing control. Blots were quantified and normalized to β-actin and presented as percentage of the control. (B) Immunofluorescence microscopy showed increased presence of TRIP6 in the focal adhesions of VSMCs expressing zyxin-shRNA, but no significant change was observed in the focal adhesion presence of paxillin and vinculin. Control and Zyxin-silenced samples were run on the same immunoblot and were from the same film exposure. The results are representative of three independent experiments. Scale bars, 30 μm.

    Techniques Used: Expressing, Western Blot, Stable Transfection, shRNA, Immunofluorescence, Microscopy

    3) Product Images from "Clostridium perfringens TpeL Glycosylates the Rac and Ras Subfamily Proteins ▿"

    Article Title: Clostridium perfringens TpeL Glycosylates the Rac and Ras Subfamily Proteins ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01019-10

    Glycosyltransferase activity of TpeL1-525. Influence of various nucleotide-hexoses on the TpeL1-525-induced glycosylation of small GTPases. Ha-Ras or Rac1 was incubated with TpeL1-525 (20 μg/ml) and UDP-[ 14 C]Glc (A) or UDP-[ 14 C]GlcNAc (B), respectively, in the presence of various nucleotide-hexoses at 37°C for 120 min. Labeled proteins were analyzed by SDS-PAGE and autoradiography. The amount of glycosylation was calculated as the percentage of the untreated control using densitometric analysis of the autoradiography. One representative experiment from three is shown. (C) Acceptor amino acid. Rac1 was glycosylated with either TpeL1-525 (20 μg/ml) or TcdB (5 μg/ml) in the presence of 30 μM UDP-[ 14 C]GlcNAc (lane 1) or 30 μM UDP-[ 14 C]Glc (lane 2). For sequential glycosylation, Rac1 was glycosylated with TpeL1-525 (lane 3) in the presence of 30 μM unlabeled UDP-GlcNAc for 60 min at 37°C and then with TcdB in the presence of 30 μM UDP-[ 14 C]Glc. In lane 4, glycosylation with TcdB and 30 μM unlabeled UDP-Glc was followed by a second glycosylation with 30 μM UDP-[ 14 C]GlcNAc and TpeL1-525. Labeled proteins were analyzed by SDS-PAGE and autoradiography.
    Figure Legend Snippet: Glycosyltransferase activity of TpeL1-525. Influence of various nucleotide-hexoses on the TpeL1-525-induced glycosylation of small GTPases. Ha-Ras or Rac1 was incubated with TpeL1-525 (20 μg/ml) and UDP-[ 14 C]Glc (A) or UDP-[ 14 C]GlcNAc (B), respectively, in the presence of various nucleotide-hexoses at 37°C for 120 min. Labeled proteins were analyzed by SDS-PAGE and autoradiography. The amount of glycosylation was calculated as the percentage of the untreated control using densitometric analysis of the autoradiography. One representative experiment from three is shown. (C) Acceptor amino acid. Rac1 was glycosylated with either TpeL1-525 (20 μg/ml) or TcdB (5 μg/ml) in the presence of 30 μM UDP-[ 14 C]GlcNAc (lane 1) or 30 μM UDP-[ 14 C]Glc (lane 2). For sequential glycosylation, Rac1 was glycosylated with TpeL1-525 (lane 3) in the presence of 30 μM unlabeled UDP-GlcNAc for 60 min at 37°C and then with TcdB in the presence of 30 μM UDP-[ 14 C]Glc. In lane 4, glycosylation with TcdB and 30 μM unlabeled UDP-Glc was followed by a second glycosylation with 30 μM UDP-[ 14 C]GlcNAc and TpeL1-525. Labeled proteins were analyzed by SDS-PAGE and autoradiography.

    Techniques Used: Activity Assay, Incubation, Gas Chromatography, Labeling, SDS Page, Autoradiography

    TpeL1-525-induced glycosylation of Rac1 in Vero cells. (A) Vero cells were incubated with SLO (100 ng/ml) alone or a combination of various amounts of TpeL1-525 with SLO (100 ng/ml) at 37°C for 15 min. After 120 min of resealing, the cells were washed and directly lysed in SDS-sample buffer. The level of Rac1 was analyzed by SDS-PAGE and Western blotting with the anti-Rac1 antibodies Mab102 and Mab23A8. (B) Signal intensities from immunoblots were recorded densitometrically. For quantification, Rac1 signals applying the glycosylation-sensitive antibody Rac1(Mab102) were normalized to the Rac1 signals applying the antibody Rac1 (Mab23A8). The Rac1 level of untreated cells was set to 1. The data shown represent the mean of three independent experiments.
    Figure Legend Snippet: TpeL1-525-induced glycosylation of Rac1 in Vero cells. (A) Vero cells were incubated with SLO (100 ng/ml) alone or a combination of various amounts of TpeL1-525 with SLO (100 ng/ml) at 37°C for 15 min. After 120 min of resealing, the cells were washed and directly lysed in SDS-sample buffer. The level of Rac1 was analyzed by SDS-PAGE and Western blotting with the anti-Rac1 antibodies Mab102 and Mab23A8. (B) Signal intensities from immunoblots were recorded densitometrically. For quantification, Rac1 signals applying the glycosylation-sensitive antibody Rac1(Mab102) were normalized to the Rac1 signals applying the antibody Rac1 (Mab23A8). The Rac1 level of untreated cells was set to 1. The data shown represent the mean of three independent experiments.

    Techniques Used: Incubation, SDS Page, Western Blot

    4) Product Images from "Matrix metalloproteinase-10 is a critical effector of protein kinase C?-Par6?-mediated lung cancer"

    Article Title: Matrix metalloproteinase-10 is a critical effector of protein kinase C?-Par6?-mediated lung cancer

    Journal: Oncogene

    doi: 10.1038/onc.2008.119

    Matrix metalloproteinase-10 (MMP-10) expression in non-small cell lung cancer (NSCLC) cells is regulated through the protein kinase Cι (PKCι)–Par6α–Rac1 signaling axis. ( a ) MMP-10 expression is regulated by PKCι, Par6α and Rac1. MMP-10 mRNA abundance was determined in NT, PKCι-RNAi, Par6α-RNAi and Rac1-RNAi cells by quantitative real-time PCR (qPCR) as described in Materials and methods. Results are expressed as % NT control. Values represent the mean ± s.d.; n = 3. Asterisk (*) denotes P
    Figure Legend Snippet: Matrix metalloproteinase-10 (MMP-10) expression in non-small cell lung cancer (NSCLC) cells is regulated through the protein kinase Cι (PKCι)–Par6α–Rac1 signaling axis. ( a ) MMP-10 expression is regulated by PKCι, Par6α and Rac1. MMP-10 mRNA abundance was determined in NT, PKCι-RNAi, Par6α-RNAi and Rac1-RNAi cells by quantitative real-time PCR (qPCR) as described in Materials and methods. Results are expressed as % NT control. Values represent the mean ± s.d.; n = 3. Asterisk (*) denotes P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    5) Product Images from "Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis"

    Article Title: Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.90590.2008

    IL-1β and transforming growth factor (TGF)-β1 in BAL are not regulated by Rac1 . Mice were exposed to 100 μg of chrysotile asbestos, and BAL was performed 1 and 21 days later. BAL fluid was analyzed for IL-1β ( A ) and TGF-β1 ( B ) by ELISA. Values, normalized to BAL total protein, are means ± SE; n = 3 animals per group.
    Figure Legend Snippet: IL-1β and transforming growth factor (TGF)-β1 in BAL are not regulated by Rac1 . Mice were exposed to 100 μg of chrysotile asbestos, and BAL was performed 1 and 21 days later. BAL fluid was analyzed for IL-1β ( A ) and TGF-β1 ( B ) by ELISA. Values, normalized to BAL total protein, are means ± SE; n = 3 animals per group.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Rac1 activation is necessary for asbestos-induced TNF-α production. A and B : WT mouse monocytes and THP-1 cells were infected for 48 h with Ad5.CMV containing either empty vector or dominant-negative N17-Rac1 vector at 500 multiplicity of infection (moi). For the last 24 h, cells were exposed to chrysotile asbestos (10 μg/cm 2 ). Conditioned medium was analyzed for TNF-α by ELISA. Values are means ± SE; n = 2. *Significantly different from chrysotile(−) Empty. **Significantly different from chrysotile(+) Empty. C : BAL cells were collected from WT and Rac1 null mice 1 and 21 days after saline or chrysotile asbestos exposure, and TNF-α mRNA was determined by real-time PCR. Values are means ± SE; n = 2. *Significantly different from WT 1 day. D : Rac1 null monocytes were infected for 48 h with Ad5.CMV containing either empty vector or Ad5.V12-Rac1 vector at 500 moi, and TNF-α mRNA was determined by real-time PCR. Values are means ± SE; n = 2. *Significantly different from Empty.
    Figure Legend Snippet: Rac1 activation is necessary for asbestos-induced TNF-α production. A and B : WT mouse monocytes and THP-1 cells were infected for 48 h with Ad5.CMV containing either empty vector or dominant-negative N17-Rac1 vector at 500 multiplicity of infection (moi). For the last 24 h, cells were exposed to chrysotile asbestos (10 μg/cm 2 ). Conditioned medium was analyzed for TNF-α by ELISA. Values are means ± SE; n = 2. *Significantly different from chrysotile(−) Empty. **Significantly different from chrysotile(+) Empty. C : BAL cells were collected from WT and Rac1 null mice 1 and 21 days after saline or chrysotile asbestos exposure, and TNF-α mRNA was determined by real-time PCR. Values are means ± SE; n = 2. *Significantly different from WT 1 day. D : Rac1 null monocytes were infected for 48 h with Ad5.CMV containing either empty vector or Ad5.V12-Rac1 vector at 500 moi, and TNF-α mRNA was determined by real-time PCR. Values are means ± SE; n = 2. *Significantly different from Empty.

    Techniques Used: Activation Assay, Infection, Plasmid Preparation, Dominant Negative Mutation, Enzyme-linked Immunosorbent Assay, Mouse Assay, Real-time Polymerase Chain Reaction

    Rac1 null mice are protected from developing asbestos-induced pulmonary fibrosis. TiO 2 ( A and B ) or chrysotile asbestos ( C and D ) was administered intratracheally at 100 μg in 50 μl of normal saline to wild-type (WT) and Rac1 null C57BL/6 mice, and animals were euthanized 21 days later. Lungs were removed and processed for collagen deposition using Masson's trichrome stain. Representative micrographs of 1 of 5 animals are shown. Scale bars, 200 μm. E : 1 and 21 days after chrysotile asbestos exposure, WT and Rac1 null mice were subjected to bronchoalveolar lavage (BAL), and hydroxyproline concentration was determined in BAL fluid. *Significantly different from Rac1 null at 21 days. F : equivalent amounts of protein from WT and Rac1 null mouse monocytes were separated by SDS-PAGE, and Western blot analysis was performed with Rac1 and β-actin monoclonal antibodies to determine the presence and equal loading of the proteins, respectively. G : WT and Rac1 null monocytes were exposed to chrysotile asbestos (10 μg/cm 2 ) for 0–60 min. Activated Rac1 was determined by binding of Rac1 to PAK-PBD beads immobilized in a 96-well plate using GLISA. Rac1 activation is expressed as relative light units (RLU) at each time point normalized to control values. H : Rac1 protein levels and equal loading of proteins in lysates from each time point in F were determined by Western blot analysis using Rac1 and β-actin monoclonal antibodies, respectively.
    Figure Legend Snippet: Rac1 null mice are protected from developing asbestos-induced pulmonary fibrosis. TiO 2 ( A and B ) or chrysotile asbestos ( C and D ) was administered intratracheally at 100 μg in 50 μl of normal saline to wild-type (WT) and Rac1 null C57BL/6 mice, and animals were euthanized 21 days later. Lungs were removed and processed for collagen deposition using Masson's trichrome stain. Representative micrographs of 1 of 5 animals are shown. Scale bars, 200 μm. E : 1 and 21 days after chrysotile asbestos exposure, WT and Rac1 null mice were subjected to bronchoalveolar lavage (BAL), and hydroxyproline concentration was determined in BAL fluid. *Significantly different from Rac1 null at 21 days. F : equivalent amounts of protein from WT and Rac1 null mouse monocytes were separated by SDS-PAGE, and Western blot analysis was performed with Rac1 and β-actin monoclonal antibodies to determine the presence and equal loading of the proteins, respectively. G : WT and Rac1 null monocytes were exposed to chrysotile asbestos (10 μg/cm 2 ) for 0–60 min. Activated Rac1 was determined by binding of Rac1 to PAK-PBD beads immobilized in a 96-well plate using GLISA. Rac1 activation is expressed as relative light units (RLU) at each time point normalized to control values. H : Rac1 protein levels and equal loading of proteins in lysates from each time point in F were determined by Western blot analysis using Rac1 and β-actin monoclonal antibodies, respectively.

    Techniques Used: Mouse Assay, Staining, Concentration Assay, SDS Page, Western Blot, Binding Assay, Activation Assay

    Rac1 activation is necessary for asbestos-induced generation of reactive oxygen species (ROS). Dichlorofluorescin diacetate (DCFH-DA, 20 μM) was added to cells during the last 20 min, and intracellular ROS levels were determined by counting the number of cells in which DCFH was oxidized to its fluorescent analog DCF by flow cytometry. A : WT and Rac1 null monocytes were exposed to chrysotile asbestos (10 μg/cm 2 ) for 24 h, and ROS generation was determined by measurement of cells labeled with DCF by flow cytometry. Values are means ± SE of relative fluorescence of DCF; n = 3 wells per treatment. *Significantly different from chrysotile(−) WT. **Significantly different from chrysotile(+) WT. B : Rac1 null monocytes were infected with Ad5.CMV containing either empty vector or constitutive active V12-Rac1 vector at 500 moi. After 24 h, cells were exposed to chrysotile asbestos (10 μg/cm 2 ) for 24 h, and ROS generation was determined as described in A . Values are means ± SE; n = 3. *Significantly different from Empty. C : WT monocytes were infected for 48 h with Ad5.CMV containing either empty vector or a catalase vector at 500 moi. Cells were exposed to chrysotile asbestos (10 μg/cm 2 ) for the last 24 h, and ROS generation was determined as described in A . Values are means ± SE; n = 3. *Significantly different from chrysotile(−) Empty. **Significantly different from chrysotile(+) Catalase.
    Figure Legend Snippet: Rac1 activation is necessary for asbestos-induced generation of reactive oxygen species (ROS). Dichlorofluorescin diacetate (DCFH-DA, 20 μM) was added to cells during the last 20 min, and intracellular ROS levels were determined by counting the number of cells in which DCFH was oxidized to its fluorescent analog DCF by flow cytometry. A : WT and Rac1 null monocytes were exposed to chrysotile asbestos (10 μg/cm 2 ) for 24 h, and ROS generation was determined by measurement of cells labeled with DCF by flow cytometry. Values are means ± SE of relative fluorescence of DCF; n = 3 wells per treatment. *Significantly different from chrysotile(−) WT. **Significantly different from chrysotile(+) WT. B : Rac1 null monocytes were infected with Ad5.CMV containing either empty vector or constitutive active V12-Rac1 vector at 500 moi. After 24 h, cells were exposed to chrysotile asbestos (10 μg/cm 2 ) for 24 h, and ROS generation was determined as described in A . Values are means ± SE; n = 3. *Significantly different from Empty. C : WT monocytes were infected for 48 h with Ad5.CMV containing either empty vector or a catalase vector at 500 moi. Cells were exposed to chrysotile asbestos (10 μg/cm 2 ) for the last 24 h, and ROS generation was determined as described in A . Values are means ± SE; n = 3. *Significantly different from chrysotile(−) Empty. **Significantly different from chrysotile(+) Catalase.

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Labeling, Fluorescence, Infection, Plasmid Preparation

    BAL cell counts ( A ) and cell differential ( B ) are similar in WT and Rac1 null mice after asbestos exposure. WT or Rac1 null mice were exposed to 100 μg of chrysotile asbestos in 50 μl of saline. After 1 and 21 days, animals were euthanized, and BAL was performed. BAL cells were counted to determine total cell count and cell differential was determined with Wright-Giemsa stain. Values are means ± SE; n = 3 animals per group. Mono, mononuclear; PMN, polymorphonuclear; Lymph, lymphocytes.
    Figure Legend Snippet: BAL cell counts ( A ) and cell differential ( B ) are similar in WT and Rac1 null mice after asbestos exposure. WT or Rac1 null mice were exposed to 100 μg of chrysotile asbestos in 50 μl of saline. After 1 and 21 days, animals were euthanized, and BAL was performed. BAL cells were counted to determine total cell count and cell differential was determined with Wright-Giemsa stain. Values are means ± SE; n = 3 animals per group. Mono, mononuclear; PMN, polymorphonuclear; Lymph, lymphocytes.

    Techniques Used: Mouse Assay, Cell Counting, Giemsa Stain

    6) Product Images from "5d, a novel analogue of 3-n-butylphthalide, decreases NADPH oxidase activity through the positive regulation of CK2 after ischemia/reperfusion injury"

    Article Title: 5d, a novel analogue of 3-n-butylphthalide, decreases NADPH oxidase activity through the positive regulation of CK2 after ischemia/reperfusion injury

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8548

    Effects of 5d on the relationship between CK2 and Rac1 protein levels Brain tissue lysates of the Sham or Mode group were immunoprecipitated with an anti-CK2α antibody A. or an anti-Rac1 antibody B. followed by Western blots with an anti-Rac1 antibody and an anti-CK2α antibody ( n = 4). C. Double labeling with an anti-CK2α antibody and an anti-Rac1 antibody using tissue lysates from brains of the Sham or Mode group ( n = 6 per group).
    Figure Legend Snippet: Effects of 5d on the relationship between CK2 and Rac1 protein levels Brain tissue lysates of the Sham or Mode group were immunoprecipitated with an anti-CK2α antibody A. or an anti-Rac1 antibody B. followed by Western blots with an anti-Rac1 antibody and an anti-CK2α antibody ( n = 4). C. Double labeling with an anti-CK2α antibody and an anti-Rac1 antibody using tissue lysates from brains of the Sham or Mode group ( n = 6 per group).

    Techniques Used: Immunoprecipitation, Western Blot, Labeling

    Effects of 5d on the translocation of cytosolic p47phox and Rac1 to membrane A. Representative Western blots of p47phox and Rac1 protein expression in membrane fractions from the brains of rats. K-Na ATPase antibody was applied as a membrane marker. B. The expression of p47phox and Rac1 protein in the membrane were normalized to K-Na ATPase level. C. Rac1 activity was measured by co-IP using GST-PAK1 PBD, followed by Western blots with a Rac1 antibody ( n = 6 per group). Data are expressed as means ± SD ( n = 4). * P
    Figure Legend Snippet: Effects of 5d on the translocation of cytosolic p47phox and Rac1 to membrane A. Representative Western blots of p47phox and Rac1 protein expression in membrane fractions from the brains of rats. K-Na ATPase antibody was applied as a membrane marker. B. The expression of p47phox and Rac1 protein in the membrane were normalized to K-Na ATPase level. C. Rac1 activity was measured by co-IP using GST-PAK1 PBD, followed by Western blots with a Rac1 antibody ( n = 6 per group). Data are expressed as means ± SD ( n = 4). * P

    Techniques Used: Translocation Assay, Western Blot, Expressing, Marker, Activity Assay, Co-Immunoprecipitation Assay

    7) Product Images from "Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis"

    Article Title: Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.055566

    Daam1 does not regulate the actin cytoskeleton through modulating small GTPase (RhoA, Rac1 and Cdc42) activities. ( A ) The expression of Dvl2 in E16.5 hearts. ( B ) Expression and phosphorylation of substrate proteins of small GTPases in E16.5 hearts. ( C ) Small GTPase activation assays. GTP-bound RhoA in E16.5 heart lysates was pulled down using GST-RBD and detected with RhoA-specific antibody. GTP-bound Rac1 and Cdc42 were pulled down using GST-PBD and detected with Rac1- and Cdc42-specific antibodies. RhoA, Rac1 and Cdc42 in lysates were detected by the above antibodies. GTPγS treatment served as positive controls for binding assays, and GAPDH served as a total protein loading control.
    Figure Legend Snippet: Daam1 does not regulate the actin cytoskeleton through modulating small GTPase (RhoA, Rac1 and Cdc42) activities. ( A ) The expression of Dvl2 in E16.5 hearts. ( B ) Expression and phosphorylation of substrate proteins of small GTPases in E16.5 hearts. ( C ) Small GTPase activation assays. GTP-bound RhoA in E16.5 heart lysates was pulled down using GST-RBD and detected with RhoA-specific antibody. GTP-bound Rac1 and Cdc42 were pulled down using GST-PBD and detected with Rac1- and Cdc42-specific antibodies. RhoA, Rac1 and Cdc42 in lysates were detected by the above antibodies. GTPγS treatment served as positive controls for binding assays, and GAPDH served as a total protein loading control.

    Techniques Used: Expressing, Activation Assay, Binding Assay

    8) Product Images from "Bone marrow‐derived mesenchymal stem cells promote invasiveness and transendothelial migration of osteosarcoma cells via a mesenchymal to amoeboid transition"

    Article Title: Bone marrow‐derived mesenchymal stem cells promote invasiveness and transendothelial migration of osteosarcoma cells via a mesenchymal to amoeboid transition

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12189

    Cross‐talk between BM ‐ MSC s and OS cells promotes the acquisition of an amoeboid‐like motility in cancer cells. (A) Gelatin zymography of CM obtained from Sa OS ‐2, MG ‐63 and HOS cells stimulated or not stimulated for 48 h with CM derived from BM ‐ MSC s activated by each OS cell line ( CM BM ‐ MSC s OS ). The white line indicates the junction of two different gels. Image is representative of three independent experiments. (B) Representative images of pull‐down assay of Rac1 and RhoA GTP ases (left panel) and related quantification (right panel). The assay was performed on OS cells grown for 48 h in CM from tumour‐activated BM ‐ MSC s ( CM BM ‐ MSC s OS ) or in starvation medium. Rac1‐ GTP and RhoA‐ GTP expression was normalized with respect to total Rac1 and RhoA in OS lysates. ### P
    Figure Legend Snippet: Cross‐talk between BM ‐ MSC s and OS cells promotes the acquisition of an amoeboid‐like motility in cancer cells. (A) Gelatin zymography of CM obtained from Sa OS ‐2, MG ‐63 and HOS cells stimulated or not stimulated for 48 h with CM derived from BM ‐ MSC s activated by each OS cell line ( CM BM ‐ MSC s OS ). The white line indicates the junction of two different gels. Image is representative of three independent experiments. (B) Representative images of pull‐down assay of Rac1 and RhoA GTP ases (left panel) and related quantification (right panel). The assay was performed on OS cells grown for 48 h in CM from tumour‐activated BM ‐ MSC s ( CM BM ‐ MSC s OS ) or in starvation medium. Rac1‐ GTP and RhoA‐ GTP expression was normalized with respect to total Rac1 and RhoA in OS lysates. ### P

    Techniques Used: Zymography, Derivative Assay, Pull Down Assay, Expressing

    9) Product Images from "Increased flux through the mevalonate pathway mediates fibrotic repair without injury"

    Article Title: Increased flux through the mevalonate pathway mediates fibrotic repair without injury

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI127959

    IPF BAL cells have reduced GGDP levels. ( A ) Schematic diagram of the mevalonate pathway. Representative mass spectrometric chromatography of FDP and GGDP in ( B ) normal ( n = 7) and ( C ) IPF ( n = 8) BAL cells. Levels of ( D ) FDP and ( E ) GGDP in BAL cells from healthy subjects ( n = 7) and patients with IPF ( n = 8) determined by mass spectrometry. ( F ) Representative immunoblot analysis of BAL cells from healthy subjects ( n = 4) and patients with IPF ( n = 5). Quantification of immunoblot in F of expression of ( G ) GGDPS and ( H ) GGTase 1 in BAL cells from healthy subjects ( n = 9–11) and patients with IPF ( n = 11). ( I ) Representative immunoblot analysis of MDMs from WT mice s.c. treated with vehicle or GGOH 10 days after exposure to saline or bleomycin ( n = 5/group). Quantification of ( J ) GGDPS and ( K ) GGTase 1 expression in I ( n = 5/group). ( L ) Acetyl-CoA concentration ( n = 4–5). ( M ) OCR and ( N ) acetyl-CoA concentration in BAL cells isolated from WT or Rac1 –/– Lyz2-Cre mice exposed to saline or bleomycin for 21 days ( n = 5 mice/group). Inset in N shows Rac1 immunoblot analysis. ( O ) OCR in transfected MH-S cells ( n = 3–4). FCCP, ; Oligo, oligomycin; FCCP, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Rot/Anti A, rotenone/antimycin A. ( P ) Acetyl-CoA concentration and ( Q ) Rac1 activity in THP-1 cells expressing scrambled siRNA or siPGC-1α together with empty or Rac1 WT and treated with GGOH ( n = 3/group). Inset in P shows PGC-1α immunoblot analysis. ( R ) Pearson’s correlation of acetyl-CoA to Rac1 activity. Values indicate the mean ± SEM. * P
    Figure Legend Snippet: IPF BAL cells have reduced GGDP levels. ( A ) Schematic diagram of the mevalonate pathway. Representative mass spectrometric chromatography of FDP and GGDP in ( B ) normal ( n = 7) and ( C ) IPF ( n = 8) BAL cells. Levels of ( D ) FDP and ( E ) GGDP in BAL cells from healthy subjects ( n = 7) and patients with IPF ( n = 8) determined by mass spectrometry. ( F ) Representative immunoblot analysis of BAL cells from healthy subjects ( n = 4) and patients with IPF ( n = 5). Quantification of immunoblot in F of expression of ( G ) GGDPS and ( H ) GGTase 1 in BAL cells from healthy subjects ( n = 9–11) and patients with IPF ( n = 11). ( I ) Representative immunoblot analysis of MDMs from WT mice s.c. treated with vehicle or GGOH 10 days after exposure to saline or bleomycin ( n = 5/group). Quantification of ( J ) GGDPS and ( K ) GGTase 1 expression in I ( n = 5/group). ( L ) Acetyl-CoA concentration ( n = 4–5). ( M ) OCR and ( N ) acetyl-CoA concentration in BAL cells isolated from WT or Rac1 –/– Lyz2-Cre mice exposed to saline or bleomycin for 21 days ( n = 5 mice/group). Inset in N shows Rac1 immunoblot analysis. ( O ) OCR in transfected MH-S cells ( n = 3–4). FCCP, ; Oligo, oligomycin; FCCP, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Rot/Anti A, rotenone/antimycin A. ( P ) Acetyl-CoA concentration and ( Q ) Rac1 activity in THP-1 cells expressing scrambled siRNA or siPGC-1α together with empty or Rac1 WT and treated with GGOH ( n = 3/group). Inset in P shows PGC-1α immunoblot analysis. ( R ) Pearson’s correlation of acetyl-CoA to Rac1 activity. Values indicate the mean ± SEM. * P

    Techniques Used: Chromatography, Mass Spectrometry, Expressing, Mouse Assay, Concentration Assay, Isolation, Transfection, Activity Assay, Pyrolysis Gas Chromatography

    Fibroblast differentiation and collagen production are macrophage dependent. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted, and lungs were extracted 11 days later. Cell suspensions from WT mice were incubated with IgG or CD11b and Ly6C antibodies. ( A ) Representative histograms of sorted cells ( n = 4/group). The percentage indicates cells removed for each condition. ( B ) Representative phase contrast microscopy pulmosphere images after a 16-hour incubation ( n = 4/group). Scale bars: 200 µm. ( C ) Percentage of the ZOI ( n = 4/group). ZOI percentage = [(total area after invasion – inner core of pulmosphere)/inner core of pulmosphere] × 100. ( D ) Immunoblot analysis of fibroblasts cultured in BALF from exposed WT and Rac1 –/– Lyz2-Cre mice ( n = 2–3 mice/group). ( E ) RhoA activity in fibroblasts cultured in BALF from exposed WT and Rac1 –/– Lyz2-Cre mice ( n = 3 mice/group). ( F ) RhoA activity in primary mouse lung fibroblasts isolated from vehicle- or GGOH-treated mice ( n = 8–10). ( G ) RhoA activity in IMR-90 fibroblasts ( n = 3). IMR-90 fibroblasts were cultured in conditioned media from vehicle- or GGOH-treated MH-S cells containing IgG control or TGF-β1–neutralizing antibody. ( H ) RhoA activity in IMR-90 fibroblasts ( n = 6/group). ( I ) Immunoblot analysis in IMR-90 fibroblasts. mRNA analysis of ( J ) Fn1 ( n = 3/group) and ( K ) Col1a1 mRNA in IMR-90s ( n = 3/group). IMR-90 fibroblasts cultured in BALF from saline- or bleomycin-exposed mice that received vehicle or GGOH. BALF was preincubated with IgG or neutralized with a TGF-β1 antibody. ( L ) RhoA activity in IMR-90 fibroblasts ( n = 3/group). ( M ) Immunoblot analysis of IMR-90 fibroblasts. Values indicate the mean ± SEM. ** P
    Figure Legend Snippet: Fibroblast differentiation and collagen production are macrophage dependent. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted, and lungs were extracted 11 days later. Cell suspensions from WT mice were incubated with IgG or CD11b and Ly6C antibodies. ( A ) Representative histograms of sorted cells ( n = 4/group). The percentage indicates cells removed for each condition. ( B ) Representative phase contrast microscopy pulmosphere images after a 16-hour incubation ( n = 4/group). Scale bars: 200 µm. ( C ) Percentage of the ZOI ( n = 4/group). ZOI percentage = [(total area after invasion – inner core of pulmosphere)/inner core of pulmosphere] × 100. ( D ) Immunoblot analysis of fibroblasts cultured in BALF from exposed WT and Rac1 –/– Lyz2-Cre mice ( n = 2–3 mice/group). ( E ) RhoA activity in fibroblasts cultured in BALF from exposed WT and Rac1 –/– Lyz2-Cre mice ( n = 3 mice/group). ( F ) RhoA activity in primary mouse lung fibroblasts isolated from vehicle- or GGOH-treated mice ( n = 8–10). ( G ) RhoA activity in IMR-90 fibroblasts ( n = 3). IMR-90 fibroblasts were cultured in conditioned media from vehicle- or GGOH-treated MH-S cells containing IgG control or TGF-β1–neutralizing antibody. ( H ) RhoA activity in IMR-90 fibroblasts ( n = 6/group). ( I ) Immunoblot analysis in IMR-90 fibroblasts. mRNA analysis of ( J ) Fn1 ( n = 3/group) and ( K ) Col1a1 mRNA in IMR-90s ( n = 3/group). IMR-90 fibroblasts cultured in BALF from saline- or bleomycin-exposed mice that received vehicle or GGOH. BALF was preincubated with IgG or neutralized with a TGF-β1 antibody. ( L ) RhoA activity in IMR-90 fibroblasts ( n = 3/group). ( M ) Immunoblot analysis of IMR-90 fibroblasts. Values indicate the mean ± SEM. ** P

    Techniques Used: Mouse Assay, Incubation, Microscopy, Cell Culture, Activity Assay, Isolation

    Increasing Rac1 activity by augmentation of isoprenylation promotes lung fibrosis. ( A ) Schematic diagram of the mevalonate pathway. HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MVADP, mevalonate 5-diphosphate. ( B ) Immunoblot analysis and ( C ) quantification of isolated mitochondria from THP-1 cells expressing empty control or Rac1 WT and treated with vehicle or GGOH (50 μM) ( n = 3). ( D ) Mitochondrial Rac1 activity in transfected MH-S cells treated with vehicle or GGOH ( n = 3). ( E ) Immunoblot analysis of transfected macrophages expressing empty control or Rac1 WT and treated with vehicle or GGOH. Cells were separated into aqueous (unprenylated) or detergent (prenylated) fractions. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were implanted s.c., and the mice were sacrificed 11 days later. ( F ) Mitochondrial Rac1 immunoblot analysis of isolated MDMs. ( G ) Isoprenylation status of Rac1 in isolated MDMs. ( H ) Mitochondrial Rac1 activity ( n = 5/group). ( I ) Tgfb1 mRNA expression (saline, vehicle n = 4; saline, GGOH n = 6; bleomycin, vehicle n = 6; bleomycin, GGOH n = 4). ( J ) Active TGF-β1 and ( K ) Ym-1 expression in BALF ( n = 5/group). ( L ) Representative lung histology images with Masson’s trichrome staining ( n = 5/group). Original magnification, ×2.5. ( M ) Hydroxyproline content ( n = 5/group). Values indicate the mean ± SEM. * P
    Figure Legend Snippet: Increasing Rac1 activity by augmentation of isoprenylation promotes lung fibrosis. ( A ) Schematic diagram of the mevalonate pathway. HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MVADP, mevalonate 5-diphosphate. ( B ) Immunoblot analysis and ( C ) quantification of isolated mitochondria from THP-1 cells expressing empty control or Rac1 WT and treated with vehicle or GGOH (50 μM) ( n = 3). ( D ) Mitochondrial Rac1 activity in transfected MH-S cells treated with vehicle or GGOH ( n = 3). ( E ) Immunoblot analysis of transfected macrophages expressing empty control or Rac1 WT and treated with vehicle or GGOH. Cells were separated into aqueous (unprenylated) or detergent (prenylated) fractions. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were implanted s.c., and the mice were sacrificed 11 days later. ( F ) Mitochondrial Rac1 immunoblot analysis of isolated MDMs. ( G ) Isoprenylation status of Rac1 in isolated MDMs. ( H ) Mitochondrial Rac1 activity ( n = 5/group). ( I ) Tgfb1 mRNA expression (saline, vehicle n = 4; saline, GGOH n = 6; bleomycin, vehicle n = 6; bleomycin, GGOH n = 4). ( J ) Active TGF-β1 and ( K ) Ym-1 expression in BALF ( n = 5/group). ( L ) Representative lung histology images with Masson’s trichrome staining ( n = 5/group). Original magnification, ×2.5. ( M ) Hydroxyproline content ( n = 5/group). Values indicate the mean ± SEM. * P

    Techniques Used: Activity Assay, Isolation, Expressing, Transfection, Mouse Assay, Staining

    GGOH-mediated lung fibrosis occurs in the absence of injury. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted; mice were sacrificed 11 days later. ( A ) Caspase-3 activity in AECs ( n = 5/group). ( B ) Representative images of lung IHC for SPC (green), TUNEL (red), and DAPI (blue) ( n = 4 per group) and ( C ) quantification of TUNEL + cells from B ( n = 4/group). Scale bars: 20 μm. WT mice were exposed to saline or bleomycin. Confocal imaging and total number of BAL cells stained with ( D and E ) CD11c ( n = 5–9) and ( F and G ) CD11b ( n = 5–9), together with DAPI. Scale bars: 50 μm. Ten days after exposure of WT mice to saline or bleomycin (Bleo), pumps containing vehicle or GGOH were s.c. implanted, and lungs were extracted 11 days later. ( H ) Representative flow cytometric plots of MDMs (CD45 + CD11b +/– Ly6G – CD64 + Ly6c – Siglec F lo ) and RAMs (CD45 + CD11b +/– Ly6G – CD64 + Ly6c – Siglec F hi ). FSC-A, forward scatter area. Total numbers of ( I ) MDMs and ( J ) RAMs from BAL. WT or CCR2 –/– mice were exposed and treated as described above. ( K ) Rac1 activity ( n = 4–5) and mRNA expression of ( L ) Tgfb1 ( n = 4–5), ( M ) Arg1 ( n = 4–5), ( N ) Tnf ( n = 4–5), and ( O ) Nos2 ( n = 4–5). Inset in K shows CCR2 immunoblot analysis. Values indicate the mean ± SEM. ** P
    Figure Legend Snippet: GGOH-mediated lung fibrosis occurs in the absence of injury. Ten days after exposure of WT mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted; mice were sacrificed 11 days later. ( A ) Caspase-3 activity in AECs ( n = 5/group). ( B ) Representative images of lung IHC for SPC (green), TUNEL (red), and DAPI (blue) ( n = 4 per group) and ( C ) quantification of TUNEL + cells from B ( n = 4/group). Scale bars: 20 μm. WT mice were exposed to saline or bleomycin. Confocal imaging and total number of BAL cells stained with ( D and E ) CD11c ( n = 5–9) and ( F and G ) CD11b ( n = 5–9), together with DAPI. Scale bars: 50 μm. Ten days after exposure of WT mice to saline or bleomycin (Bleo), pumps containing vehicle or GGOH were s.c. implanted, and lungs were extracted 11 days later. ( H ) Representative flow cytometric plots of MDMs (CD45 + CD11b +/– Ly6G – CD64 + Ly6c – Siglec F lo ) and RAMs (CD45 + CD11b +/– Ly6G – CD64 + Ly6c – Siglec F hi ). FSC-A, forward scatter area. Total numbers of ( I ) MDMs and ( J ) RAMs from BAL. WT or CCR2 –/– mice were exposed and treated as described above. ( K ) Rac1 activity ( n = 4–5) and mRNA expression of ( L ) Tgfb1 ( n = 4–5), ( M ) Arg1 ( n = 4–5), ( N ) Tnf ( n = 4–5), and ( O ) Nos2 ( n = 4–5). Inset in K shows CCR2 immunoblot analysis. Values indicate the mean ± SEM. ** P

    Techniques Used: Mouse Assay, Activity Assay, Immunohistochemistry, TUNEL Assay, Imaging, Staining, Flow Cytometry, Expressing

    mtROS is required for Rac1-mediated profibrotic polarization of BAL cells. mtROS generation in BAL cells from ( A ) healthy subjects ( n = 6) or patients with IPF ( n = 6) and from ( B ) saline- ( n = 6) or bleomycin-exposed ( n = 6) mice. ( C ) mtROS generation in THP-1 cells expressing empty control or Rac1 WT and treated with vehicle or GGOH ( n = 3). ( D ) Ten days after exposure of mice to saline or bleomycin, pumps containing vehicle or GGOH were implanted s.c. into mice, and the mice were sacrificed 11 days later. Data show mtROS generation in isolated MDMs ( n = 5 mice/group). ( E ) mtROS generation in isolated BAL cells from bleomycin- or saline-exposed WT or Rac1 –/– Lyz2-Cre mice (saline, n = 4 mice; bleomycin n = 6 mice). Inset shows immunoblot analysis. ( F ) Nuclear immunoblot analysis of transfected THP-1 cells treated with vehicle or GGOH ( n = 3). ( G ) mtROS generation in transfected THP-1 cells ( n = 5). Inset shows Rieske immunoblot analysis. Nuclear immunoblot analysis of THP-1 cells expressing ( H ) scrambled or Rieske siRNA and empty control or Rac1 WT ( n = 3) and ( I ) empty, Rac1 CA , or Rac1 DN . Arg1 promoter activity in transfected ( J ) THP-1 cells ( n = 3) and ( K ) MH-S cells ( n = 3). Inset in J shows KLF4 and HIF-2α immunoblot analysis. mRNA expression of ( L ) Tgfb1 ( n = 3) and ( M ) Chil3 ( n = 3) in MH-S cells expressing empty control, Rac1 WT , or Rac1 C189S . mRNA expression of ( N ) Tgfb1 ( n = 3) and ( O ) Retnla ( n = 3) in transfected THP-1 cells. Inset in N shows GGDPS immunoblot analysis. Values indicate the mean ± SEM. * P
    Figure Legend Snippet: mtROS is required for Rac1-mediated profibrotic polarization of BAL cells. mtROS generation in BAL cells from ( A ) healthy subjects ( n = 6) or patients with IPF ( n = 6) and from ( B ) saline- ( n = 6) or bleomycin-exposed ( n = 6) mice. ( C ) mtROS generation in THP-1 cells expressing empty control or Rac1 WT and treated with vehicle or GGOH ( n = 3). ( D ) Ten days after exposure of mice to saline or bleomycin, pumps containing vehicle or GGOH were implanted s.c. into mice, and the mice were sacrificed 11 days later. Data show mtROS generation in isolated MDMs ( n = 5 mice/group). ( E ) mtROS generation in isolated BAL cells from bleomycin- or saline-exposed WT or Rac1 –/– Lyz2-Cre mice (saline, n = 4 mice; bleomycin n = 6 mice). Inset shows immunoblot analysis. ( F ) Nuclear immunoblot analysis of transfected THP-1 cells treated with vehicle or GGOH ( n = 3). ( G ) mtROS generation in transfected THP-1 cells ( n = 5). Inset shows Rieske immunoblot analysis. Nuclear immunoblot analysis of THP-1 cells expressing ( H ) scrambled or Rieske siRNA and empty control or Rac1 WT ( n = 3) and ( I ) empty, Rac1 CA , or Rac1 DN . Arg1 promoter activity in transfected ( J ) THP-1 cells ( n = 3) and ( K ) MH-S cells ( n = 3). Inset in J shows KLF4 and HIF-2α immunoblot analysis. mRNA expression of ( L ) Tgfb1 ( n = 3) and ( M ) Chil3 ( n = 3) in MH-S cells expressing empty control, Rac1 WT , or Rac1 C189S . mRNA expression of ( N ) Tgfb1 ( n = 3) and ( O ) Retnla ( n = 3) in transfected THP-1 cells. Inset in N shows GGDPS immunoblot analysis. Values indicate the mean ± SEM. * P

    Techniques Used: Mouse Assay, Expressing, Isolation, Transfection, Activity Assay

    Rac1 is required for GGOH- and bleomycin-induced lung fibrosis. Ten days after exposure of WT and Rac1 –/– Lyz2-Cre mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted, and the mice were sacrificed 11 days later. ( A ) Total number of BAL cells and ( B ) cell differential ( n = 5/group). Mac, macrophage; PMN, polymorphonuclear. ( C ) Mitochondrial Rac1 activity ( n = 5/group). Inset in C shows Rac1 immunoblot analysis of BAL cells. ( D ) mtROS generation in isolated BAL cells ( n = 5–6/group). ( E ) Active TGF-β1, ( F ) PDGF-BB, and ( G ) Ym-1 expression in BALF ( n = 4–5/group). ( H and I ) Ten days after exposure of WT mice to saline or bleomycin, daily i.p. injections of simvastatin (SIMV) (20 mg/kg/day) were administered, and mice were sacrificed 11 days later. ( H ) Active TGF-β1 and ( I ) PDGF-BB expression in BALF ( n = 4/group). ( J ) Representative images of lung histology with Masson’s trichrome staining ( n = 5/group). Original magnification, ×2.5. ( K ) Hydroxyproline content ( n = 5/group). Values indicate the mean ± SEM. ** P
    Figure Legend Snippet: Rac1 is required for GGOH- and bleomycin-induced lung fibrosis. Ten days after exposure of WT and Rac1 –/– Lyz2-Cre mice to saline or bleomycin, pumps containing vehicle or GGOH were s.c. implanted, and the mice were sacrificed 11 days later. ( A ) Total number of BAL cells and ( B ) cell differential ( n = 5/group). Mac, macrophage; PMN, polymorphonuclear. ( C ) Mitochondrial Rac1 activity ( n = 5/group). Inset in C shows Rac1 immunoblot analysis of BAL cells. ( D ) mtROS generation in isolated BAL cells ( n = 5–6/group). ( E ) Active TGF-β1, ( F ) PDGF-BB, and ( G ) Ym-1 expression in BALF ( n = 4–5/group). ( H and I ) Ten days after exposure of WT mice to saline or bleomycin, daily i.p. injections of simvastatin (SIMV) (20 mg/kg/day) were administered, and mice were sacrificed 11 days later. ( H ) Active TGF-β1 and ( I ) PDGF-BB expression in BALF ( n = 4/group). ( J ) Representative images of lung histology with Masson’s trichrome staining ( n = 5/group). Original magnification, ×2.5. ( K ) Hydroxyproline content ( n = 5/group). Values indicate the mean ± SEM. ** P

    Techniques Used: Mouse Assay, Activity Assay, Isolation, Expressing, Staining

    Mitochondrial Rac1 activity is increased in IPF BAL cells. Immunoblot analysis of Rac1 expression in isolated ( A ) mitochondria or ( B ) cytosol fractions from BAL cells obtained from healthy subjects ( n = 4) or patients with IPF ( n = 4). ( C ) Mitochondrial Rac1 activity in BAL cells from healthy subjects ( n = 6) or patients with IPF ( n = 5). BAL cells from healthy subjects ( n = 6–7) or patients with IPF ( n = 6–8) were analyzed for mRNA expression of ( D ) ARG1 and ( E ) PDGFB . ( F ) Arginase 1 activity in BAL cells from healthy subjects ( n = 5) or patients with IPF ( n = 8). ( G ) Rac1 activity in BAL cells isolated at the indicated time points from mice exposed to saline or bleomycin ( n = 3–4 mice/time point). Immunoblot analysis of Rac1 expression in isolated ( H ) mitochondria or ( I ) cytosolic fractions from BAL cells obtained from mice treated with saline ( n = 4) or bleomycin ( n = 5). ( J ) Mitochondrial Rac1 activity in BAL cells from saline- ( n = 5) or bleomycin-exposed ( n = 5) mice. ( K ) PDGF-BB and ( L ) IL-10 were measured in BALF from saline- or bleomycin-exposed mice at the indicated time points ( n = 3–4 mice/time point). Values indicate the mean ± SEM. * P
    Figure Legend Snippet: Mitochondrial Rac1 activity is increased in IPF BAL cells. Immunoblot analysis of Rac1 expression in isolated ( A ) mitochondria or ( B ) cytosol fractions from BAL cells obtained from healthy subjects ( n = 4) or patients with IPF ( n = 4). ( C ) Mitochondrial Rac1 activity in BAL cells from healthy subjects ( n = 6) or patients with IPF ( n = 5). BAL cells from healthy subjects ( n = 6–7) or patients with IPF ( n = 6–8) were analyzed for mRNA expression of ( D ) ARG1 and ( E ) PDGFB . ( F ) Arginase 1 activity in BAL cells from healthy subjects ( n = 5) or patients with IPF ( n = 8). ( G ) Rac1 activity in BAL cells isolated at the indicated time points from mice exposed to saline or bleomycin ( n = 3–4 mice/time point). Immunoblot analysis of Rac1 expression in isolated ( H ) mitochondria or ( I ) cytosolic fractions from BAL cells obtained from mice treated with saline ( n = 4) or bleomycin ( n = 5). ( J ) Mitochondrial Rac1 activity in BAL cells from saline- ( n = 5) or bleomycin-exposed ( n = 5) mice. ( K ) PDGF-BB and ( L ) IL-10 were measured in BALF from saline- or bleomycin-exposed mice at the indicated time points ( n = 3–4 mice/time point). Values indicate the mean ± SEM. * P

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

    10) Product Images from "Antroquinonol Targets FAK-Signaling Pathway Suppressed Cell Migration, Invasion, and Tumor Growth of C6 Glioma"

    Article Title: Antroquinonol Targets FAK-Signaling Pathway Suppressed Cell Migration, Invasion, and Tumor Growth of C6 Glioma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141285

    Effects of antroquinonol on FAK signaling pathway. Cells were treated with the indicated concentrations of antroquinonol (5 and 10 μM) for 24 h. Cells were harvested and lysed for western blot analysis. (A) Changes in the levels of FAK, pFAK, Src, pSrc, Cdc42, and Rac1 proteins after normalization to the levels of beta-actin are shown below each blot. (B) The in vitro assay was performed to detect the effect of antroquinonol on pFAK (Y397) (i) N18 and (ii) C6. (C) Cells were treated with PF 431396 (0.5 and 2 μM) for 24 h. Proteins were extracted and subjected to western blot analysis for FAK and pFAK. The results shown are the mean ±SEM of three independent experiments. * p
    Figure Legend Snippet: Effects of antroquinonol on FAK signaling pathway. Cells were treated with the indicated concentrations of antroquinonol (5 and 10 μM) for 24 h. Cells were harvested and lysed for western blot analysis. (A) Changes in the levels of FAK, pFAK, Src, pSrc, Cdc42, and Rac1 proteins after normalization to the levels of beta-actin are shown below each blot. (B) The in vitro assay was performed to detect the effect of antroquinonol on pFAK (Y397) (i) N18 and (ii) C6. (C) Cells were treated with PF 431396 (0.5 and 2 μM) for 24 h. Proteins were extracted and subjected to western blot analysis for FAK and pFAK. The results shown are the mean ±SEM of three independent experiments. * p

    Techniques Used: Western Blot, In Vitro

    11) Product Images from "Brain-derived Neurotrophic Factor (BDNF) Induces Polarized Signaling of Small GTPase (Rac1) Protein at the Onset of Schwann Cell Myelination through Partitioning-defective 3 (Par3) Protein"

    Article Title: Brain-derived Neurotrophic Factor (BDNF) Induces Polarized Signaling of Small GTPase (Rac1) Protein at the Onset of Schwann Cell Myelination through Partitioning-defective 3 (Par3) Protein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.312736

    Par3 is necessary for BDNF-mediated but not for NRG1-Type III-mediated Rac1 activation in Schwann cell cultures. A , Par3 is necessary for BDNF-mediated Rac1 activation in Schwann cell cultures. Following infection with the retrovirus carrying Par3 RNAi
    Figure Legend Snippet: Par3 is necessary for BDNF-mediated but not for NRG1-Type III-mediated Rac1 activation in Schwann cell cultures. A , Par3 is necessary for BDNF-mediated Rac1 activation in Schwann cell cultures. Following infection with the retrovirus carrying Par3 RNAi

    Techniques Used: Activation Assay, Infection

    BDNF and NRG1-Type III activate Rac1 in sciatic nerves. A , RacGTP levels are their highest at E19.5 in rats and gradually decline during development. B, quantification of RacGTP levels in A. n = 6–8. C , asymmetric localization of phospho-PAK immunoreactivity
    Figure Legend Snippet: BDNF and NRG1-Type III activate Rac1 in sciatic nerves. A , RacGTP levels are their highest at E19.5 in rats and gradually decline during development. B, quantification of RacGTP levels in A. n = 6–8. C , asymmetric localization of phospho-PAK immunoreactivity

    Techniques Used:

    P75 signals from Schwann cells activate Rac1 and promote myelination. A , knockdown of p75 in rat Schwann cells inhibited the extent of myelination in culture. Also shown are the control Westerns for the p75 knockdown with RNAi. For statistical analyses,
    Figure Legend Snippet: P75 signals from Schwann cells activate Rac1 and promote myelination. A , knockdown of p75 in rat Schwann cells inhibited the extent of myelination in culture. Also shown are the control Westerns for the p75 knockdown with RNAi. For statistical analyses,

    Techniques Used:

    Selective knockdown of Par3 among Schwann cells in vivo results in disruption of polarized Rac1 activation and attenuation in myelination. A , representative images of the retrovirus-infected sciatic nerves. GFP signals indicate the circular myelin sheath
    Figure Legend Snippet: Selective knockdown of Par3 among Schwann cells in vivo results in disruption of polarized Rac1 activation and attenuation in myelination. A , representative images of the retrovirus-infected sciatic nerves. GFP signals indicate the circular myelin sheath

    Techniques Used: In Vivo, Activation Assay, Infection

    12) Product Images from "The Repetitive Oligopeptide Sequences Modulate Cytopathic Potency but Are Not Crucial for Cellular Uptake of Clostridium difficile Toxin A"

    Article Title: The Repetitive Oligopeptide Sequences Modulate Cytopathic Potency but Are Not Crucial for Cellular Uptake of Clostridium difficile Toxin A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017623

    Cytotoxic potency of TcdA and TcdA 1–1874 towards host cells. A) HT29 cells were treated with full length TcdA or TcdA 1–1874 in a concentration-dependent manner until onset of cell rounding. Western blot analysis was performed to monitor level of glucosylated Rac1 using antibodies recognizing either non-glucosylated Rac1 (upper panel) or total Rac1 (lower panel), respectively. B) Dose-dependent analysis of cytopathic effect (CPE) and Rac1-glucosylation induced by TcdA (•) and TcdA 1–1874 (▿) on 3T3, HT29 and CHO-C6 cells. Cytopathic effect was quantified as round cells per total cells in %. Results of Rac1-glucosylation are based on immunoblot analyses exemplarily shown in A). Values are given as means ± SD, n = 3.
    Figure Legend Snippet: Cytotoxic potency of TcdA and TcdA 1–1874 towards host cells. A) HT29 cells were treated with full length TcdA or TcdA 1–1874 in a concentration-dependent manner until onset of cell rounding. Western blot analysis was performed to monitor level of glucosylated Rac1 using antibodies recognizing either non-glucosylated Rac1 (upper panel) or total Rac1 (lower panel), respectively. B) Dose-dependent analysis of cytopathic effect (CPE) and Rac1-glucosylation induced by TcdA (•) and TcdA 1–1874 (▿) on 3T3, HT29 and CHO-C6 cells. Cytopathic effect was quantified as round cells per total cells in %. Results of Rac1-glucosylation are based on immunoblot analyses exemplarily shown in A). Values are given as means ± SD, n = 3.

    Techniques Used: Concentration Assay, Western Blot

    TcdA 1–1874 lacking the C-terminal repeats still possesses cytotoxic potency. A) Multidomain structure of C. difficile TcdA and TcdA mutants TcdA 1–1874 and TcdA 1–1101 . Full length TcdA consists of the N-terminal glucosyltransferase domain (GTD), the cysteinprotease domain (CPD), the hydrophobic region (HR) acting as transmembrane domain and the C-terminal combined repetitive oligopeptides (CROPs). The CROPs were deleted in TcdA 1–1874 . Mutant TcdA 1–1101 exhibits the whole N-terminal domain including the hydrophobic region. B) Cell rounding assay of 3T3 fibroblasts after 90 min of toxin treatment with 1 nM of TcdA, TcdA 1–1874 or TcdA 1–1101 , respectively. C) Western blot analysis of toxin-treated cells using antibody either recognizing only non-glucosylated (upper panel) or total Rac1 (lower panel). D) Pre-treatment of 3T3 fibroblasts with 100 nM of Bafilomycin A1, an inhibitor of endosomal acidification, prevents cell rounding of TcdA and TcdA 1–1874 revealing specific cellular uptake of CROP-truncated TcdA. Cells were treated with equipotent concentrations of TcdA (1 nM) and TcdA 1–1874 (10 nM) for 2 h.
    Figure Legend Snippet: TcdA 1–1874 lacking the C-terminal repeats still possesses cytotoxic potency. A) Multidomain structure of C. difficile TcdA and TcdA mutants TcdA 1–1874 and TcdA 1–1101 . Full length TcdA consists of the N-terminal glucosyltransferase domain (GTD), the cysteinprotease domain (CPD), the hydrophobic region (HR) acting as transmembrane domain and the C-terminal combined repetitive oligopeptides (CROPs). The CROPs were deleted in TcdA 1–1874 . Mutant TcdA 1–1101 exhibits the whole N-terminal domain including the hydrophobic region. B) Cell rounding assay of 3T3 fibroblasts after 90 min of toxin treatment with 1 nM of TcdA, TcdA 1–1874 or TcdA 1–1101 , respectively. C) Western blot analysis of toxin-treated cells using antibody either recognizing only non-glucosylated (upper panel) or total Rac1 (lower panel). D) Pre-treatment of 3T3 fibroblasts with 100 nM of Bafilomycin A1, an inhibitor of endosomal acidification, prevents cell rounding of TcdA and TcdA 1–1874 revealing specific cellular uptake of CROP-truncated TcdA. Cells were treated with equipotent concentrations of TcdA (1 nM) and TcdA 1–1874 (10 nM) for 2 h.

    Techniques Used: Mutagenesis, Western Blot

    13) Product Images from "Ovarian cancer G protein coupled receptor 1 suppresses cell migration of MCF7 breast cancer cells via a G?12/13-Rho-Rac1 pathway"

    Article Title: Ovarian cancer G protein coupled receptor 1 suppresses cell migration of MCF7 breast cancer cells via a G?12/13-Rho-Rac1 pathway

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-8-6

    Effects of OGR1 over-expression on the activity of Rho family members in MCF7 cells. The activation levels of Rho ( A ), Rac1 ( B ) and Cdc42 ( C ) were examined by pull-down and Western blot analyses as described in Materials and Methods. Total Rho, Rac1, and Cdc42, as well as α-tubulin were analyzed in whole cell lysates. Representative results are from three independent experiments.
    Figure Legend Snippet: Effects of OGR1 over-expression on the activity of Rho family members in MCF7 cells. The activation levels of Rho ( A ), Rac1 ( B ) and Cdc42 ( C ) were examined by pull-down and Western blot analyses as described in Materials and Methods. Total Rho, Rac1, and Cdc42, as well as α-tubulin were analyzed in whole cell lysates. Representative results are from three independent experiments.

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

    OGR1 inhibited MCF7 breast cancer cell migration through a Gα 12/13 -Rho-Rac1 pathway. Cells were pretreated with the solvent or PTX (1 μM) for 16 h, or transfected with the RGS plasmid for 48 h. ( A ) Cell migration was analyzed by transwell migration assays after treatment or using transient transfected cells 72 h post-transfection. *** P
    Figure Legend Snippet: OGR1 inhibited MCF7 breast cancer cell migration through a Gα 12/13 -Rho-Rac1 pathway. Cells were pretreated with the solvent or PTX (1 μM) for 16 h, or transfected with the RGS plasmid for 48 h. ( A ) Cell migration was analyzed by transwell migration assays after treatment or using transient transfected cells 72 h post-transfection. *** P

    Techniques Used: Migration, Transfection, Plasmid Preparation

    14) Product Images from "The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation"

    Article Title: The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00418.2012

    Rac1 associates with a set of PAK effectors from thrombin-activated platelets.
    Figure Legend Snippet: Rac1 associates with a set of PAK effectors from thrombin-activated platelets.

    Techniques Used:

    15) Product Images from "20-HETE increases Superoxide production and activates NADPH Oxidase in Pulmonary Artery Endothelial Cells"

    Article Title: 20-HETE increases Superoxide production and activates NADPH Oxidase in Pulmonary Artery Endothelial Cells

    Journal:

    doi: 10.1152/ajplung.00278.2007

    20-HETE increases Rac1/2 in BPAECs
    Figure Legend Snippet: 20-HETE increases Rac1/2 in BPAECs

    Techniques Used:

    16) Product Images from "Regulation of neurite outgrowth by Gi/o signaling pathways"

    Article Title: Regulation of neurite outgrowth by Gi/o signaling pathways

    Journal:

    doi:

    G i/o signaling to the actin cytoskeleton during the induction of neurite outgrowth. Signaling from the G i/o -coupled CB1R to its effectors GRIN, Cdc42 and Rac1 and their potential downstream targets is shown in the schematic. The intermediate molecules
    Figure Legend Snippet: G i/o signaling to the actin cytoskeleton during the induction of neurite outgrowth. Signaling from the G i/o -coupled CB1R to its effectors GRIN, Cdc42 and Rac1 and their potential downstream targets is shown in the schematic. The intermediate molecules

    Techniques Used:

    17) Product Images from "Rac Activation by the T-Cell Receptor Inhibits T Cell Migration"

    Article Title: Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012393

    A phosphomimetic moesin mutant reverts Rac1-L61 inhibition of T cell polarization and motility. (A) Cells transfected with the indicated plasmids were stained for ICAM-3 and the HA tag on moesin. Moe-TD-HA, moesin-T558D-HA; Moe-TA-HA, moesin-T558A-HA. Representative images are shown. Scale bar, 10 µm. (B) Frequency of each cell morphology; mean of the % of cells in 3 independent experiments +/− S.E.M. Only transfected cells were analysed. Polarized: cells with uropod and clustering of ICAM-3 (open bars). Non-polarized: Cells with multiple protrusions or absence of uropod and homogeneously distributed ICAM-3 (black bars). (C) T cells expressing the indicated proteins were imaged by time-lapse microscopy on ICAM-1; migration speed of transfected cells pre-treated with the indicated antibodies is the mean of the average speed in four different experiments +/− S.E.M.; p
    Figure Legend Snippet: A phosphomimetic moesin mutant reverts Rac1-L61 inhibition of T cell polarization and motility. (A) Cells transfected with the indicated plasmids were stained for ICAM-3 and the HA tag on moesin. Moe-TD-HA, moesin-T558D-HA; Moe-TA-HA, moesin-T558A-HA. Representative images are shown. Scale bar, 10 µm. (B) Frequency of each cell morphology; mean of the % of cells in 3 independent experiments +/− S.E.M. Only transfected cells were analysed. Polarized: cells with uropod and clustering of ICAM-3 (open bars). Non-polarized: Cells with multiple protrusions or absence of uropod and homogeneously distributed ICAM-3 (black bars). (C) T cells expressing the indicated proteins were imaged by time-lapse microscopy on ICAM-1; migration speed of transfected cells pre-treated with the indicated antibodies is the mean of the average speed in four different experiments +/− S.E.M.; p

    Techniques Used: Mutagenesis, Inhibition, Transfection, Staining, Expressing, Time-lapse Microscopy, Migration

    Rac activation is required for TCR-induced inhibition of migration and polarity. (A) T cells expressing GFP or GFP-Rac1-L61 (constitutively active) were assayed for migration in ICAM-1-coated transwells. The mean of 3 independent experiments +/− S.E.M. is shown; *p
    Figure Legend Snippet: Rac activation is required for TCR-induced inhibition of migration and polarity. (A) T cells expressing GFP or GFP-Rac1-L61 (constitutively active) were assayed for migration in ICAM-1-coated transwells. The mean of 3 independent experiments +/− S.E.M. is shown; *p

    Techniques Used: Activation Assay, Inhibition, Migration, Expressing

    Regulation of Rho GTPase activity and localization by TCR activation. (A) T cells were starved for 5 h and incubated in anti-CD3 or control IgG antibody-coated wells. After 45 min cells were lysed and RhoA, Rac1 and Cdc42 activity assayed in pull-down experiments. A representative experiment out of at least 3 for each Rho GTPase is shown. Graphs show active Rho GTPase (GTP) levels:total levels as mean of 5 experiments (RhoA and Rac1) or 3 experiments (Cdc42) +/− S.E.M. relative to IgG pre-treated cells. *p
    Figure Legend Snippet: Regulation of Rho GTPase activity and localization by TCR activation. (A) T cells were starved for 5 h and incubated in anti-CD3 or control IgG antibody-coated wells. After 45 min cells were lysed and RhoA, Rac1 and Cdc42 activity assayed in pull-down experiments. A representative experiment out of at least 3 for each Rho GTPase is shown. Graphs show active Rho GTPase (GTP) levels:total levels as mean of 5 experiments (RhoA and Rac1) or 3 experiments (Cdc42) +/− S.E.M. relative to IgG pre-treated cells. *p

    Techniques Used: Activity Assay, Activation Assay, Incubation

    Schematic model for mechanisms leading to TCR-induced loss of migratory polarity. TCR engagement by antigen leads to activation of Rac1 (increased Rac1-GTP) and inhibition of RhoA (decreased RhoA-GTP). Active Rac1 stimulates stathmin phosphorylation, leading to microtubule stabilization, and reduces ERM phosphorylation, leading to loss of uropod structures. Active RhoA normally stimulates ERM phosphorylation and reduces microtubule stability, and these responses are reduced when RhoA activity decreases. Loss of the uropod together with increased microtubule stability reduces migratory polarity and hence inhibits migration.
    Figure Legend Snippet: Schematic model for mechanisms leading to TCR-induced loss of migratory polarity. TCR engagement by antigen leads to activation of Rac1 (increased Rac1-GTP) and inhibition of RhoA (decreased RhoA-GTP). Active Rac1 stimulates stathmin phosphorylation, leading to microtubule stabilization, and reduces ERM phosphorylation, leading to loss of uropod structures. Active RhoA normally stimulates ERM phosphorylation and reduces microtubule stability, and these responses are reduced when RhoA activity decreases. Loss of the uropod together with increased microtubule stability reduces migratory polarity and hence inhibits migration.

    Techniques Used: Activation Assay, Inhibition, Activity Assay, Migration

    18) Product Images from "SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline"

    Article Title: SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8786

    Proposed mechanism demonstrating the interaction between the lipid-raft associated Orai1/TRPC1/SK3 channel complex and EGFR signaling pathway in colon cancer cell migration This model suggests three positive feedback loops: 1) STIM1 following its phosphorylation by EGF stimulation and Akt, is the trigger of SOCE and promotes migration mediated by the translocation of TRPC1 and Orai1 into lipid rafts where SK3 is concentrated. 2) The Orai1/TRPC1/SK3 channel complex promotes SOCE, which enhances P-Akt leading to the phosphorylation of STIM1, that may promote SOCE and 3) P-Akt activates Rac1, which enhances SOCE and in turn P-Akt. These loops operated toward the same goal: enhancing SOCE and SK3-dependent cell migration.
    Figure Legend Snippet: Proposed mechanism demonstrating the interaction between the lipid-raft associated Orai1/TRPC1/SK3 channel complex and EGFR signaling pathway in colon cancer cell migration This model suggests three positive feedback loops: 1) STIM1 following its phosphorylation by EGF stimulation and Akt, is the trigger of SOCE and promotes migration mediated by the translocation of TRPC1 and Orai1 into lipid rafts where SK3 is concentrated. 2) The Orai1/TRPC1/SK3 channel complex promotes SOCE, which enhances P-Akt leading to the phosphorylation of STIM1, that may promote SOCE and 3) P-Akt activates Rac1, which enhances SOCE and in turn P-Akt. These loops operated toward the same goal: enhancing SOCE and SK3-dependent cell migration.

    Techniques Used: Migration, Translocation Assay

    Rac1 basal activity promotes SOCE-dependent migration through the activation of the downstream effectors PI3K/Akt A. PI3K and Akt are involved in SOCE. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated for 1h with LY294002, a potent inhibitor of PI3K (upper panel,) or with MK2206, an inhibitor of Akt (lower panel). Data represent means ± SEM. *p
    Figure Legend Snippet: Rac1 basal activity promotes SOCE-dependent migration through the activation of the downstream effectors PI3K/Akt A. PI3K and Akt are involved in SOCE. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated for 1h with LY294002, a potent inhibitor of PI3K (upper panel,) or with MK2206, an inhibitor of Akt (lower panel). Data represent means ± SEM. *p

    Techniques Used: Activity Assay, Migration, Activation Assay, Fluorescence

    PI3K/Akt/Rac1 signaling pathway mediates EGF-induced SOCE dependent cell migration via P-STIM1 A. Increased of SOCE induced by EGF is inhibited by PD153035. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated for 20min with EGF +/− PD153035. Results are expressed as mean ± SEM. ***p
    Figure Legend Snippet: PI3K/Akt/Rac1 signaling pathway mediates EGF-induced SOCE dependent cell migration via P-STIM1 A. Increased of SOCE induced by EGF is inhibited by PD153035. Fluorescence measurement and relative fluorescence of Ca 2+ entry after intracellular calcium store depletion by Tg in cells treated for 20min with EGF +/− PD153035. Results are expressed as mean ± SEM. ***p

    Techniques Used: Migration, Fluorescence

    19) Product Images from "The role of Sdf-1? signaling in Xenopus laevis somite morphogenesis"

    Article Title: The role of Sdf-1? signaling in Xenopus laevis somite morphogenesis

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.24092

    RhoA and Rac1 activation through sdf-1α signaling pathway
    Figure Legend Snippet: RhoA and Rac1 activation through sdf-1α signaling pathway

    Techniques Used: Activation Assay

    20) Product Images from "Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-?-catenin and Wnt-RhoA-Rac1 pathways"

    Article Title: Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-?-catenin and Wnt-RhoA-Rac1 pathways

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200806049

    Recruitment of Dvl by BMP-2 requires Smads and is independent of BMPRII functional status. (A) Active RhoA and Rac1 pull-down experiments on hPAEC nucleofected with either vector or dominant-negative (Δ) Smad1 construct and incubated with 10 ng/ml BMP-2 were analyzed as described in Fig. 6 . **, P
    Figure Legend Snippet: Recruitment of Dvl by BMP-2 requires Smads and is independent of BMPRII functional status. (A) Active RhoA and Rac1 pull-down experiments on hPAEC nucleofected with either vector or dominant-negative (Δ) Smad1 construct and incubated with 10 ng/ml BMP-2 were analyzed as described in Fig. 6 . **, P

    Techniques Used: Functional Assay, Plasmid Preparation, Dominant Negative Mutation, Construct, Incubation

    BMP-2 and Wnt3a increase levels of active RhoA and Rac1 in hPAECs. Active RhoA and Rac1 pull-down experiments were performed on hPAECs starved for 24 h followed by incubation with 10 ng/ml BMP-2 or 100 ng/ml Wnt3a for 1 and 4 h. Representative immunoblots for RhoA and Rac1 are shown along with densitometry. Levels of active RhoA and Rac1 were measured against total RhoA and Rac1 in cell lysates. Error bars denote mean ± SEM for three different experiments with triplicate assessments. *, P
    Figure Legend Snippet: BMP-2 and Wnt3a increase levels of active RhoA and Rac1 in hPAECs. Active RhoA and Rac1 pull-down experiments were performed on hPAECs starved for 24 h followed by incubation with 10 ng/ml BMP-2 or 100 ng/ml Wnt3a for 1 and 4 h. Representative immunoblots for RhoA and Rac1 are shown along with densitometry. Levels of active RhoA and Rac1 were measured against total RhoA and Rac1 in cell lysates. Error bars denote mean ± SEM for three different experiments with triplicate assessments. *, P

    Techniques Used: Incubation, Western Blot

    Schema illustrating that regulation of proliferation, survival, and migration in hPAECs depends on cross talk between BMP and Wnt pathways. (A) BMP-2 and Wnt3a promote an increase in β-C–mediated transcriptional activity and up-regulate gene targets involved in proliferation and survival. (B) However, migration is the result of a Smad1 or, with reduced BMPRII, a Smad3-dependent recruitment of Dvl, which allows selective activation of RhoA, Rac1, and downward targets involved with cytoskeletal reorganization and cell motility.
    Figure Legend Snippet: Schema illustrating that regulation of proliferation, survival, and migration in hPAECs depends on cross talk between BMP and Wnt pathways. (A) BMP-2 and Wnt3a promote an increase in β-C–mediated transcriptional activity and up-regulate gene targets involved in proliferation and survival. (B) However, migration is the result of a Smad1 or, with reduced BMPRII, a Smad3-dependent recruitment of Dvl, which allows selective activation of RhoA, Rac1, and downward targets involved with cytoskeletal reorganization and cell motility.

    Techniques Used: Migration, Activity Assay, Activation Assay

    BMP-2 recruits the PDZ and DEP domains of Dvl to activate RhoA and Rac1 and induce hPAEC motility. (A) Diagram illustrating the structure of the four Dvl-GFP constructs used in the experiments described in this study. (B and C) Dvl constructs illustrated in A were individually nucleofected in hPAECs to assess impact on RhoA (B) and Rac1 (C) activation in the presence of 10 ng/ml BMP-2. Pull-down experiments were performed and analyzed as in Fig. 5 . Error bars denote mean ± SEM for three different experiments with triplicate assessments. *, P
    Figure Legend Snippet: BMP-2 recruits the PDZ and DEP domains of Dvl to activate RhoA and Rac1 and induce hPAEC motility. (A) Diagram illustrating the structure of the four Dvl-GFP constructs used in the experiments described in this study. (B and C) Dvl constructs illustrated in A were individually nucleofected in hPAECs to assess impact on RhoA (B) and Rac1 (C) activation in the presence of 10 ng/ml BMP-2. Pull-down experiments were performed and analyzed as in Fig. 5 . Error bars denote mean ± SEM for three different experiments with triplicate assessments. *, P

    Techniques Used: Construct, Activation Assay

    21) Product Images from "Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src"

    Article Title: Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src

    Journal: Cytoskeleton (Hoboken, N.J.)

    doi: 10.1002/cm.21181

    Rac1 and PIP5-kinase are not sufficient for ventral actin structures and actin and membrane dynamics in HeLa cells treated with PMA
    Figure Legend Snippet: Rac1 and PIP5-kinase are not sufficient for ventral actin structures and actin and membrane dynamics in HeLa cells treated with PMA

    Techniques Used:

    22) Product Images from "Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors"

    Article Title: Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors

    Journal: Haematologica

    doi: 10.3324/haematol.2016.147694

    Rho GTPase expression analysis. (A) Western blot analysis and quantification of CDC42, RAC1 and RHOA expression in platelet lysates from healthy controls (n=7 for CDC42, n=4 for RAC1, n=4 for RHOA) and affected members from F1 (n=6 for CDC42, n=4 for
    Figure Legend Snippet: Rho GTPase expression analysis. (A) Western blot analysis and quantification of CDC42, RAC1 and RHOA expression in platelet lysates from healthy controls (n=7 for CDC42, n=4 for RAC1, n=4 for RHOA) and affected members from F1 (n=6 for CDC42, n=4 for

    Techniques Used: Expressing, Western Blot

    23) Product Images from "Activation of adenosine receptor A2A increases HSC proliferation and inhibits death and senescence by down-regulation of p53 and Rb"

    Article Title: Activation of adenosine receptor A2A increases HSC proliferation and inhibits death and senescence by down-regulation of p53 and Rb

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2014.00069

    A cAMP analog, or an activator of adenylyl cyclase Forskolin, or NECA down regulates p53 and Rb through down regulation of Rac1 and decreased activation of p38 MAPK in the HSC . LX-2 cells were treated for 2 days, and 10 days cultured activated primary rat HSC were treated for 5 days with 8-Bromo-cAMP (1mM) or, Forskolin (50 μM) or NECA (10 μM) in presence or absence of PKA inhibitor 12–14 amide (myristoylated; 10 μM) or H-89 dihydrochloride hydrate (25 μM). (A) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 2 days treated LX-2 cells. (B) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 5 days treated activated rat primary HSC. (C) Summary of adenosine receptor A 2A mediated signaling in the process of senescence in HSC.
    Figure Legend Snippet: A cAMP analog, or an activator of adenylyl cyclase Forskolin, or NECA down regulates p53 and Rb through down regulation of Rac1 and decreased activation of p38 MAPK in the HSC . LX-2 cells were treated for 2 days, and 10 days cultured activated primary rat HSC were treated for 5 days with 8-Bromo-cAMP (1mM) or, Forskolin (50 μM) or NECA (10 μM) in presence or absence of PKA inhibitor 12–14 amide (myristoylated; 10 μM) or H-89 dihydrochloride hydrate (25 μM). (A) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 2 days treated LX-2 cells. (B) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 5 days treated activated rat primary HSC. (C) Summary of adenosine receptor A 2A mediated signaling in the process of senescence in HSC.

    Techniques Used: Activation Assay, Cell Culture, Western Blot

    24) Product Images from "Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I"

    Article Title: Rac1 and Stathmin but Not EB1 Are Required for Invasion of Breast Cancer Cells in Response to IGF-I

    Journal: International Journal of Cell Biology

    doi: 10.1155/2011/615912

    Overexpression and activation of Rac1 by IGF-I in MDA-MB-231 but not in MCF7 cells. (a) Cells after stimulation with IGF-I for 0, 0.5, or 6 h were lysed and processed for immunoblotting with anti-Rac1 antibody. Band intensity was measured and values are given as Rac1 expression relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, total and activated Rac1 were immunoprecipitated from the cell lysates and 4- or 8-fold concentrated cell lysates, respectively, and processed for immunoblotting with anti-Rac1 antibody. Band intensity was measured and the mean (SD) values of triplicate experiments are given as the amount of active Rac1 relative to total Rac1.
    Figure Legend Snippet: Overexpression and activation of Rac1 by IGF-I in MDA-MB-231 but not in MCF7 cells. (a) Cells after stimulation with IGF-I for 0, 0.5, or 6 h were lysed and processed for immunoblotting with anti-Rac1 antibody. Band intensity was measured and values are given as Rac1 expression relative to that in unstimulated control MDA-MB-231 cells. (b) After stimulation with IGF-I, total and activated Rac1 were immunoprecipitated from the cell lysates and 4- or 8-fold concentrated cell lysates, respectively, and processed for immunoblotting with anti-Rac1 antibody. Band intensity was measured and the mean (SD) values of triplicate experiments are given as the amount of active Rac1 relative to total Rac1.

    Techniques Used: Over Expression, Activation Assay, Multiple Displacement Amplification, Expressing, Immunoprecipitation

    Rac1 and stathmin but not EB1 are required for IGF-I-induced invasion of MDA-MB-231 cells. Cells after incubation for 48 h with mismatch negative control small interfering RNA and 2 different small interfering RNAs of Rac1 (a), stathmin (b), or EB1 (c) were lysed and the same amounts of total cell lysates were immunoblotted with antibodies to β -actin and Rac1 (a), stathmin (b), or EB1 (c). Cells transfected with control small interfering RNA and Rac1-1 (a), stathmin-1 (b), or EB1-1 small interfering RNA (c) were incubated for 6 h toward medium containing (+) or lacking (−) IGF-I. The mean (SD) values of triplicate assays are given as the number of invaded cells relative to that in unstimulated control MDA-MB-231 cells.
    Figure Legend Snippet: Rac1 and stathmin but not EB1 are required for IGF-I-induced invasion of MDA-MB-231 cells. Cells after incubation for 48 h with mismatch negative control small interfering RNA and 2 different small interfering RNAs of Rac1 (a), stathmin (b), or EB1 (c) were lysed and the same amounts of total cell lysates were immunoblotted with antibodies to β -actin and Rac1 (a), stathmin (b), or EB1 (c). Cells transfected with control small interfering RNA and Rac1-1 (a), stathmin-1 (b), or EB1-1 small interfering RNA (c) were incubated for 6 h toward medium containing (+) or lacking (−) IGF-I. The mean (SD) values of triplicate assays are given as the number of invaded cells relative to that in unstimulated control MDA-MB-231 cells.

    Techniques Used: Multiple Displacement Amplification, Incubation, Negative Control, Small Interfering RNA, Transfection

    25) Product Images from "Rictor regulates cell migration by suppressing RhoGDI2"

    Article Title: Rictor regulates cell migration by suppressing RhoGDI2

    Journal: Oncogene

    doi: 10.1038/onc.2012.287

    RhoGDI2 suppresses activity of Rac and Cdc42 GTPases and inhibits cell migration in the rictor null cells. (a) The cell images indicating the cell migration efficiency of the rictor null cells with or without knock down of RhoGDI2. The transwell cell migration assay was perfomed as in Fig. 1A . (b) The diagram indicating the percentage of migrated cells shown in (a). (c) Immunoblotting of the cellular lysates and pull down of the GTP-bound Rac1 obtained from cells shown in (a) to detect abundance of RhoGDI2 and Rac1 activity.
    Figure Legend Snippet: RhoGDI2 suppresses activity of Rac and Cdc42 GTPases and inhibits cell migration in the rictor null cells. (a) The cell images indicating the cell migration efficiency of the rictor null cells with or without knock down of RhoGDI2. The transwell cell migration assay was perfomed as in Fig. 1A . (b) The diagram indicating the percentage of migrated cells shown in (a). (c) Immunoblotting of the cellular lysates and pull down of the GTP-bound Rac1 obtained from cells shown in (a) to detect abundance of RhoGDI2 and Rac1 activity.

    Techniques Used: Activity Assay, Migration, Cell Migration Assay

    26) Product Images from "Synaptic P-Rex1 signaling regulates hippocampal long-term depression and autism-like social behavior"

    Article Title: Synaptic P-Rex1 signaling regulates hippocampal long-term depression and autism-like social behavior

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

    doi: 10.1073/pnas.1512913112

    P-Rex1–mediated Rac1 activation in the CA1 region of the hippocampus is involved in NMDAR LTD. Representative samples of Western blots of the Rac1 activation assay and GluR1 (Ser845) phosphorylation without NMDA (0 min) and with NMDA (100 μM) treatment for 2, 5, and 10 min in hippocampal slices of WT and Prex1 −/− (KO) mice. A low level of Rac1 activity (GTP-Rac1 divided by total Rac1) ( A ) but a normal GluR1 (Ser845) phosphorylation level (phosphorylated divided by total) ( B ) was seen with the application of NMDA in Prex1 −/− slices ( n = 6 mice per genotype). Repeated-measures two-way ANOVA with genotype and treatment as independent variables was used for statistical comparisons. Data are presented as mean ± SEM.
    Figure Legend Snippet: P-Rex1–mediated Rac1 activation in the CA1 region of the hippocampus is involved in NMDAR LTD. Representative samples of Western blots of the Rac1 activation assay and GluR1 (Ser845) phosphorylation without NMDA (0 min) and with NMDA (100 μM) treatment for 2, 5, and 10 min in hippocampal slices of WT and Prex1 −/− (KO) mice. A low level of Rac1 activity (GTP-Rac1 divided by total Rac1) ( A ) but a normal GluR1 (Ser845) phosphorylation level (phosphorylated divided by total) ( B ) was seen with the application of NMDA in Prex1 −/− slices ( n = 6 mice per genotype). Repeated-measures two-way ANOVA with genotype and treatment as independent variables was used for statistical comparisons. Data are presented as mean ± SEM.

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

    The inhibition or recovery of P-Rex1 or Rac1 activity and the hippocampal injection of Rac activator rescued social recognition inflexibility in Prex1 −/− mice. ( A ) The hippocampal Rac1 activity was reduced by NSC23766 injection compared with vehicle (saline) injection. ( B ) Overexpression of P-Rex1 protein by LV-mediated WT human P-Rex1 in the Prex1 −/− hippocampal CA1 region 7 d before analysis. ( C ) The activation level of Rac1 was restored by AAV-mediated overexpression of WT Rac1 protein in the Prex1 −/− hippocampal CA1 region 7 d before analysis. ( D ) Western blot showing the GTP-Rac1 level in the hippocampus 90 min after the injection of Rac activator or vehicle. ( E – H ) Three-chamber social interaction test. Two pipes were buried in the bilateral hippocampus of 4-wk-old Prex1 −/− mice, and the behavior tests were performed at age 6 wk. Ninety minutes before testing, hippocampi were injected with saline or Rac activator (0.1 μg/μL, 1 μL per unilateral site). ( E and F ) In the sociability trial, both the Rac activator and the vehicle groups spent more time in the chamber containing the social partner (stranger 1) or spent more time in close interaction with stranger 1. ( G and H ) In the social novelty preference trial, the mice in the Rac activator group displayed a preference for the novel social partner (stranger 2) and spent more time in close interaction with stranger 2 than did the mice in the vehicle group ( n = 9 mice per group). ** P
    Figure Legend Snippet: The inhibition or recovery of P-Rex1 or Rac1 activity and the hippocampal injection of Rac activator rescued social recognition inflexibility in Prex1 −/− mice. ( A ) The hippocampal Rac1 activity was reduced by NSC23766 injection compared with vehicle (saline) injection. ( B ) Overexpression of P-Rex1 protein by LV-mediated WT human P-Rex1 in the Prex1 −/− hippocampal CA1 region 7 d before analysis. ( C ) The activation level of Rac1 was restored by AAV-mediated overexpression of WT Rac1 protein in the Prex1 −/− hippocampal CA1 region 7 d before analysis. ( D ) Western blot showing the GTP-Rac1 level in the hippocampus 90 min after the injection of Rac activator or vehicle. ( E – H ) Three-chamber social interaction test. Two pipes were buried in the bilateral hippocampus of 4-wk-old Prex1 −/− mice, and the behavior tests were performed at age 6 wk. Ninety minutes before testing, hippocampi were injected with saline or Rac activator (0.1 μg/μL, 1 μL per unilateral site). ( E and F ) In the sociability trial, both the Rac activator and the vehicle groups spent more time in the chamber containing the social partner (stranger 1) or spent more time in close interaction with stranger 1. ( G and H ) In the social novelty preference trial, the mice in the Rac activator group displayed a preference for the novel social partner (stranger 2) and spent more time in close interaction with stranger 2 than did the mice in the vehicle group ( n = 9 mice per group). ** P

    Techniques Used: Inhibition, Activity Assay, Injection, Mouse Assay, Over Expression, Activation Assay, Western Blot

    Interaction of PP1α with P-Rex1 and P-Rex1–mediated Rac1 activation are essential for NMDA-induced AMPAR endocytosis. ( A ) Coimmunoprecipitation of PP1α with P-Rex1 in hippocampal slices from the CA1 region treated acutely with 100 μM NMDA. ( B ) Quantification of the NMDA-induced interaction of P-Rex1 with PP1α from five independent experiments. The ratio in the WT group was defined as 1.0. ( C – I ) Hippocampal neurons expressing pCAGGS-IRES-EGFP (Veh) vector ( C and D ), WT P-Rex1 (P-Rex1-WT) ( E ), WT Rac1 (Rac1-WT) ( F ), P-Rex1 with the VAFA mutation (P-Rex1-VAFA) ( G ), and P-Rex1 with the GEF domain dead mutation (P-Rex1-DH dead) ( H ) were treated with 50 μM NMDA for 10 min and stained for surface HA-GluR2 (red). After Triton X-100 treatment, the neurons were stained for the total HA-GluR2 (blue). The dendritic regions marked by white squares are magnified in the bottom panels. (Scale bars, 10 μm.) ( I ) Quantification of the NMDA-induced endocytosis of surface GluR2. Data are presented as the ratio of the intensity of surface staining HA-GluR2 to the intensity of total HA-GluR2 staining. The ratio in control neurons was defined as 1.0. (WT+Veh: n = 12 in the control group and n = 12 in the NMDA group; KO+Veh: n = 12 in the control group and n = 14 in the NMDA group; KO+P-Rex1 WT: n = 10 in the control group and n = 11 in the NMDA group; KO+Rac1 WT: n = 11 in the control group and n = 12 in the NMDA group; KO+P-Rex1 VAFA: n = 10 in the control group and n = 11 in the NMDA group; KO+P-Rex1 DHdead: n = 12 in the control group and n = 13 in the NMDA group). * P
    Figure Legend Snippet: Interaction of PP1α with P-Rex1 and P-Rex1–mediated Rac1 activation are essential for NMDA-induced AMPAR endocytosis. ( A ) Coimmunoprecipitation of PP1α with P-Rex1 in hippocampal slices from the CA1 region treated acutely with 100 μM NMDA. ( B ) Quantification of the NMDA-induced interaction of P-Rex1 with PP1α from five independent experiments. The ratio in the WT group was defined as 1.0. ( C – I ) Hippocampal neurons expressing pCAGGS-IRES-EGFP (Veh) vector ( C and D ), WT P-Rex1 (P-Rex1-WT) ( E ), WT Rac1 (Rac1-WT) ( F ), P-Rex1 with the VAFA mutation (P-Rex1-VAFA) ( G ), and P-Rex1 with the GEF domain dead mutation (P-Rex1-DH dead) ( H ) were treated with 50 μM NMDA for 10 min and stained for surface HA-GluR2 (red). After Triton X-100 treatment, the neurons were stained for the total HA-GluR2 (blue). The dendritic regions marked by white squares are magnified in the bottom panels. (Scale bars, 10 μm.) ( I ) Quantification of the NMDA-induced endocytosis of surface GluR2. Data are presented as the ratio of the intensity of surface staining HA-GluR2 to the intensity of total HA-GluR2 staining. The ratio in control neurons was defined as 1.0. (WT+Veh: n = 12 in the control group and n = 12 in the NMDA group; KO+Veh: n = 12 in the control group and n = 14 in the NMDA group; KO+P-Rex1 WT: n = 10 in the control group and n = 11 in the NMDA group; KO+Rac1 WT: n = 11 in the control group and n = 12 in the NMDA group; KO+P-Rex1 VAFA: n = 10 in the control group and n = 11 in the NMDA group; KO+P-Rex1 DHdead: n = 12 in the control group and n = 13 in the NMDA group). * P

    Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Mutagenesis, Staining

    P-Rex1–Rac1 signaling in the CA1 region of the hippocampus plays a vital role in social recognition behavior. ( A ) LV carrying GFP was injected into the CA1 region of 3-wk-old mice and was observed after 3 wk. The GFP was expressed in different coronal sections of the hippocampus limited to the CA1 region. ( B ) Western blot showing knockdown of endogenous P-Rex1 by LV-mediated shRNA injection in the CA1 region. ( C and D ) The social recognition test was performed 3 wk after LV injection. ( C ) In the sociability trial, mice injected with saline, control shRNA, or P-Rex1 shRNA spent more time in close interaction with the social partner (stranger 1, S1) than in the empty cage (EM). ( D ) In the social novelty preference trial, mice injected with P-Rex1 shRNA did not display a preference for the novel social partner (stranger 2, S2) as compared with the saline or control shRNA group ( n = 10 mice per group). ( E and F ) The level of Rac1 activity (GTP-Rac1 divided by total Rac1), but not Cdc42 or RhoA, was decreased significantly in the hippocampi of Prex1 −/− mice. ( G and H ) Mice were injected with vehicle or with the Rac1 inhibitor NSC23766. ( G ) Both groups performed normally in the sociability trial. ( H ) In the social novelty preference trial the mice injected with NSC23766 spent almost equal time in close interaction with stranger 1 and stranger 2 ( n = 9 mice per group). * P
    Figure Legend Snippet: P-Rex1–Rac1 signaling in the CA1 region of the hippocampus plays a vital role in social recognition behavior. ( A ) LV carrying GFP was injected into the CA1 region of 3-wk-old mice and was observed after 3 wk. The GFP was expressed in different coronal sections of the hippocampus limited to the CA1 region. ( B ) Western blot showing knockdown of endogenous P-Rex1 by LV-mediated shRNA injection in the CA1 region. ( C and D ) The social recognition test was performed 3 wk after LV injection. ( C ) In the sociability trial, mice injected with saline, control shRNA, or P-Rex1 shRNA spent more time in close interaction with the social partner (stranger 1, S1) than in the empty cage (EM). ( D ) In the social novelty preference trial, mice injected with P-Rex1 shRNA did not display a preference for the novel social partner (stranger 2, S2) as compared with the saline or control shRNA group ( n = 10 mice per group). ( E and F ) The level of Rac1 activity (GTP-Rac1 divided by total Rac1), but not Cdc42 or RhoA, was decreased significantly in the hippocampi of Prex1 −/− mice. ( G and H ) Mice were injected with vehicle or with the Rac1 inhibitor NSC23766. ( G ) Both groups performed normally in the sociability trial. ( H ) In the social novelty preference trial the mice injected with NSC23766 spent almost equal time in close interaction with stranger 1 and stranger 2 ( n = 9 mice per group). * P

    Techniques Used: Injection, Mouse Assay, Western Blot, shRNA, Activity Assay

    Recovery of reversal learning in the MWM and fear extinction but not repetitive grooming in Prex1 −/− mice. ( A ) Overexpression of WT P-Rex1 made no difference in the latency in locating a hidden platform across 7 d of training compared with control group. ( B ) Probe test. Note that overexpression of WT P-Rex1 resulted in almost identical time spent in the target quadrant compared with control group. ( C ) WT P-Rex1 overexpression resulted in a clear augmentation of reversal learning in KO mice because they required less time to reach the new location of the hidden platform. ( D ) Reversal probe test. Note that group with WT P-Rex1 overexpression spent significantly more time in the new target quadrant ( n = 11 in the KO+CTL group; n = 10 in the KO+WT P-Rex1 group). ( E ) Freezing time was increased significantly after one unconditional stimulus per block in both WT P-Rex1 and control groups. ( F ) Freezing time was decreased more significantly in the group overexpressing WT P-Rex1 than in the control group 24 h after learning ( n = 13 mice per group). ( G ) Stereotypical grooming was not significantly different between the group overexpressing WT P-Rex1 and the control group ( n = 9 mice per group). ( H ) Overexpression of WT Rac1 made no difference in the latency in locating a hidden platform across 7 d of training compared with the control group. ( I ) Probe test. Note that overexpression of WT Rac1 led to almost identical time spent in the target quadrant compared with the control group. ( J ) WT Rac1 overexpression resulted in a clear augmentation of reversal learning in KO mice because they required less time to reach the new location of the platform. ( K ) Reversal probe test. Note that the group overexpressing WT Rac1 spent significantly more time than the control group in the new target quadrant ( n = 12 for the KO+CTL group; n = 11 for the KO+WT Rac1 group). ( L ) Freezing time was increased significantly in both the WT Rac1 and control groups after one unconditional stimulus per block. ( M ) Freezing time was decreased more significantly in the group overexpressing WT Rac1 than in the control group 24 h after conditioning to the stimulus ( n = 11 mice per group). ( N ) Stereotypical grooming was not significantly different between the group overexpressing WT Rac1 and the control group ( n = 10 mice per group). * P
    Figure Legend Snippet: Recovery of reversal learning in the MWM and fear extinction but not repetitive grooming in Prex1 −/− mice. ( A ) Overexpression of WT P-Rex1 made no difference in the latency in locating a hidden platform across 7 d of training compared with control group. ( B ) Probe test. Note that overexpression of WT P-Rex1 resulted in almost identical time spent in the target quadrant compared with control group. ( C ) WT P-Rex1 overexpression resulted in a clear augmentation of reversal learning in KO mice because they required less time to reach the new location of the hidden platform. ( D ) Reversal probe test. Note that group with WT P-Rex1 overexpression spent significantly more time in the new target quadrant ( n = 11 in the KO+CTL group; n = 10 in the KO+WT P-Rex1 group). ( E ) Freezing time was increased significantly after one unconditional stimulus per block in both WT P-Rex1 and control groups. ( F ) Freezing time was decreased more significantly in the group overexpressing WT P-Rex1 than in the control group 24 h after learning ( n = 13 mice per group). ( G ) Stereotypical grooming was not significantly different between the group overexpressing WT P-Rex1 and the control group ( n = 9 mice per group). ( H ) Overexpression of WT Rac1 made no difference in the latency in locating a hidden platform across 7 d of training compared with the control group. ( I ) Probe test. Note that overexpression of WT Rac1 led to almost identical time spent in the target quadrant compared with the control group. ( J ) WT Rac1 overexpression resulted in a clear augmentation of reversal learning in KO mice because they required less time to reach the new location of the platform. ( K ) Reversal probe test. Note that the group overexpressing WT Rac1 spent significantly more time than the control group in the new target quadrant ( n = 12 for the KO+CTL group; n = 11 for the KO+WT Rac1 group). ( L ) Freezing time was increased significantly in both the WT Rac1 and control groups after one unconditional stimulus per block. ( M ) Freezing time was decreased more significantly in the group overexpressing WT Rac1 than in the control group 24 h after conditioning to the stimulus ( n = 11 mice per group). ( N ) Stereotypical grooming was not significantly different between the group overexpressing WT Rac1 and the control group ( n = 10 mice per group). * P

    Techniques Used: Mouse Assay, Over Expression, CTL Assay, Blocking Assay

    27) Product Images from "Ovarian cancer G protein coupled receptor 1 suppresses cell migration of MCF7 breast cancer cells via a G?12/13-Rho-Rac1 pathway"

    Article Title: Ovarian cancer G protein coupled receptor 1 suppresses cell migration of MCF7 breast cancer cells via a G?12/13-Rho-Rac1 pathway

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-8-6

    Effects of OGR1 over-expression on the activity of Rho family members in MCF7 cells. The activation levels of Rho ( A ), Rac1 ( B ) and Cdc42 ( C ) were examined by pull-down and Western blot analyses as described in Materials and Methods. Total Rho, Rac1, and Cdc42, as well as α-tubulin were analyzed in whole cell lysates. Representative results are from three independent experiments.
    Figure Legend Snippet: Effects of OGR1 over-expression on the activity of Rho family members in MCF7 cells. The activation levels of Rho ( A ), Rac1 ( B ) and Cdc42 ( C ) were examined by pull-down and Western blot analyses as described in Materials and Methods. Total Rho, Rac1, and Cdc42, as well as α-tubulin were analyzed in whole cell lysates. Representative results are from three independent experiments.

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

    OGR1 inhibited MCF7 breast cancer cell migration through a Gα 12/13 -Rho-Rac1 pathway. Cells were pretreated with the solvent or PTX (1 μM) for 16 h, or transfected with the RGS plasmid for 48 h. ( A ) Cell migration was analyzed by transwell migration assays after treatment or using transient transfected cells 72 h post-transfection. *** P
    Figure Legend Snippet: OGR1 inhibited MCF7 breast cancer cell migration through a Gα 12/13 -Rho-Rac1 pathway. Cells were pretreated with the solvent or PTX (1 μM) for 16 h, or transfected with the RGS plasmid for 48 h. ( A ) Cell migration was analyzed by transwell migration assays after treatment or using transient transfected cells 72 h post-transfection. *** P

    Techniques Used: Migration, Transfection, Plasmid Preparation

    28) Product Images from "Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C, Focal Adhesion Kinase, and Ezrin to Promote Traversal across the Blood-Brain Barrier *"

    Article Title: Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C, Focal Adhesion Kinase, and Ezrin to Promote Traversal across the Blood-Brain Barrier *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.389676

    Current model of the host signaling events involved in cryptococcal traversal of the BBB. Cryptococcus binding to the host receptor(s) on HBMEC triggers activation of RhoGTPases such as RhoA, Rac1, and Cdc42. Subsequently, active RhoGTPases induces phosphorylation of other host signaling proteins regulating actin cytoskeleton. Both Rac1 and Cdc42 are involved in phosphorylation of FAK, ezrin, and PKCα, whereas RhoA leads to phosphorylation of FAK and ezrin but not PKCα. Activation of those host proteins results in the host actin cytoskeleton rearrangement and formation of microvilli-like membrane protrusions associated with invading Cryptococcus cells followed by the cryptococcal invasion and transmigration across the HBMEC monolayer. CD44 has shown to play a role as a host cellular receptor for hyaluronic acid of Cryptococcus . However, it is not fully understood how Cryptococcus -CD44 interaction triggers activation of host proteins yet.
    Figure Legend Snippet: Current model of the host signaling events involved in cryptococcal traversal of the BBB. Cryptococcus binding to the host receptor(s) on HBMEC triggers activation of RhoGTPases such as RhoA, Rac1, and Cdc42. Subsequently, active RhoGTPases induces phosphorylation of other host signaling proteins regulating actin cytoskeleton. Both Rac1 and Cdc42 are involved in phosphorylation of FAK, ezrin, and PKCα, whereas RhoA leads to phosphorylation of FAK and ezrin but not PKCα. Activation of those host proteins results in the host actin cytoskeleton rearrangement and formation of microvilli-like membrane protrusions associated with invading Cryptococcus cells followed by the cryptococcal invasion and transmigration across the HBMEC monolayer. CD44 has shown to play a role as a host cellular receptor for hyaluronic acid of Cryptococcus . However, it is not fully understood how Cryptococcus -CD44 interaction triggers activation of host proteins yet.

    Techniques Used: Binding Assay, Activation Assay, Transmigration Assay

    C. neoformans induces activation of RhoGTPases in HBMEC required for their transmigration. A , confluent HBMEC monolayers were incubated with C. neoformans (1 × 10 6 ) for the indicated times, and Rho pulldown assays were performed to determine activation of RhoA, Rac1, and Cdc42. The level of activation was determined by densitometry analysis (measuring the ratios of GTP-Rho/total Rho). B , HBMEC was transduced with adenoviral constructs of DN RhoGTPases mutants (N19RhoA, N17Rac1 or N17Cdc42). Expression of DN RhoGTPases in the transduced HBMEC was verified by Western blotting with anti-myc antibody ( bottom ). Transmigration assay with HBMEC expressing DN RhoGTPases were carried out. These experiments were repeated three times independently. *, p
    Figure Legend Snippet: C. neoformans induces activation of RhoGTPases in HBMEC required for their transmigration. A , confluent HBMEC monolayers were incubated with C. neoformans (1 × 10 6 ) for the indicated times, and Rho pulldown assays were performed to determine activation of RhoA, Rac1, and Cdc42. The level of activation was determined by densitometry analysis (measuring the ratios of GTP-Rho/total Rho). B , HBMEC was transduced with adenoviral constructs of DN RhoGTPases mutants (N19RhoA, N17Rac1 or N17Cdc42). Expression of DN RhoGTPases in the transduced HBMEC was verified by Western blotting with anti-myc antibody ( bottom ). Transmigration assay with HBMEC expressing DN RhoGTPases were carried out. These experiments were repeated three times independently. *, p

    Techniques Used: Activation Assay, Transmigration Assay, Incubation, Transduction, Construct, Expressing, Western Blot

    29) Product Images from "The WASF3-NCKAP1-CYFIP1 complex is essential for breast cancer metastasis"

    Article Title: The WASF3-NCKAP1-CYFIP1 complex is essential for breast cancer metastasis

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-0562

    RAC1 binding to the WASF3 complex is required for NCKAP1-mediated invasion of breast cancer cells
    Figure Legend Snippet: RAC1 binding to the WASF3 complex is required for NCKAP1-mediated invasion of breast cancer cells

    Techniques Used: Binding Assay

    30) Product Images from "Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42"

    Article Title: Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003276

    IL-6 stimulation leads to activation of CDC42 in pancreatic cancer cells. A , IL-6 rapidly activates CDC42. PANC-1 cells were treated with IL-6 for the indicated times (50 ng/ml), then lysed, and analyzed for CDC42 activation using a GST-PBD biochemical pulldown and Western blotting for CDC42. Note the rapid and robust activation of CDC42 within just 5 min post-stimulation. Active CDC42 was normalized to total CDC42 and compared with the ratio at t = 0. B , in contrast, IL-6 stimulation did not activate RAC1. PANC-1 cells were treated as described in A , and active RAC1 was precipitated using a GST-PBD pulldown followed by Western blotting for RAC1. Active RAC1 was normalized to total RAC1 and compared with the ratio at t = 0. C , IL-6 does not significantly activate RhoA. PANC-1 cells were stimulated with IL-6 as above, and active RhoA was precipitated using a GST-RBD pulldown followed by Western blotting for RhoA. Active RhoA was normalized to total RhoA and compared with the ratio at t = 0. Relative levels of RAC1, CDC42, or RhoA activation are graphed as the mean of three to six independent biological replicates. Error bars represent S.E. * indicates p .
    Figure Legend Snippet: IL-6 stimulation leads to activation of CDC42 in pancreatic cancer cells. A , IL-6 rapidly activates CDC42. PANC-1 cells were treated with IL-6 for the indicated times (50 ng/ml), then lysed, and analyzed for CDC42 activation using a GST-PBD biochemical pulldown and Western blotting for CDC42. Note the rapid and robust activation of CDC42 within just 5 min post-stimulation. Active CDC42 was normalized to total CDC42 and compared with the ratio at t = 0. B , in contrast, IL-6 stimulation did not activate RAC1. PANC-1 cells were treated as described in A , and active RAC1 was precipitated using a GST-PBD pulldown followed by Western blotting for RAC1. Active RAC1 was normalized to total RAC1 and compared with the ratio at t = 0. C , IL-6 does not significantly activate RhoA. PANC-1 cells were stimulated with IL-6 as above, and active RhoA was precipitated using a GST-RBD pulldown followed by Western blotting for RhoA. Active RhoA was normalized to total RhoA and compared with the ratio at t = 0. Relative levels of RAC1, CDC42, or RhoA activation are graphed as the mean of three to six independent biological replicates. Error bars represent S.E. * indicates p .

    Techniques Used: Activation Assay, Western Blot

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    Incubation:

    Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling
    Article Snippet: .. After blocking, the samples were incubated with primary antibodies: rabbit anti-GFP (Invitrogen), mouse or rabbit anti-HA (Covance; Abcam), mouse anti-Rac1 (EMD Millipore), goat or rabbit anti-PlexinD1 (R & D Systems; a gift from Y. Yoshida), and goat anti-SH3BP1 (Everest Biotech Ltd.) overnight at 4°C. .. Cells were washed three times for 5 min in 1× PBS and incubated with Alexa Fluor 488 anti–rabbit and Alexa Fluor 567 anti–mouse secondary antibodies for 1 h at room temperature.

    other:

    Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes
    Article Snippet: The following antibody dilutions were used: rabbit anti-Trio 1:100–1:1000; mouse anti-Rac1: 1:1000; mouse anti-p-p38 1:1000; mouse anti-p38 1:1000; rabbit anti-CdGAP 1:1000; rabbit anti-βPIX 1:1000; mouse anti-tubulin 1:5000; rabbit anti-calnexin 1:1000.

    Immunofluorescence:

    Article Title: Nonpolarized signaling reveals two distinct modes of 3D cell migration
    Article Snippet: .. Mouse anti-Rac1 (Millipore) and rabbit anti-Cdc42 (Abcam) were used for immunofluorescence analysis of endogenous GTPases. .. To localize endogenous Rho family GTPases, PIP2, or PIP3, HFFs were fixed and stained following a published protocol for preservation of the plasma membrane during immunocytochemistry ( ) but with slight modifications.

    Blocking Assay:

    Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling
    Article Snippet: .. After blocking, the samples were incubated with primary antibodies: rabbit anti-GFP (Invitrogen), mouse or rabbit anti-HA (Covance; Abcam), mouse anti-Rac1 (EMD Millipore), goat or rabbit anti-PlexinD1 (R & D Systems; a gift from Y. Yoshida), and goat anti-SH3BP1 (Everest Biotech Ltd.) overnight at 4°C. .. Cells were washed three times for 5 min in 1× PBS and incubated with Alexa Fluor 488 anti–rabbit and Alexa Fluor 567 anti–mouse secondary antibodies for 1 h at room temperature.

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  • 85
    Millipore myc rac1
    Effect of AA and SDS on the interaction of the p67 phox ·Rac-GTP complex with Nox2. A , SDS-PAGE analysis of purified proteins used in this study. GST alone, GST-Nox2-C, the wild-type p67 phox <t>(1–212)-Rac1</t> (Q61L) chimera, and a mutant p67
    Myc Rac1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore gtp rac1 pull down assay
    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of <t>GTP</t> (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of <t>Rac1</t> GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value
    Gtp Rac1 Pull Down Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of AA and SDS on the interaction of the p67 phox ·Rac-GTP complex with Nox2. A , SDS-PAGE analysis of purified proteins used in this study. GST alone, GST-Nox2-C, the wild-type p67 phox (1–212)-Rac1 (Q61L) chimera, and a mutant p67

    Journal: The Journal of Biological Chemistry

    Article Title: Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production *

    doi: 10.1074/jbc.M114.581785

    Figure Lengend Snippet: Effect of AA and SDS on the interaction of the p67 phox ·Rac-GTP complex with Nox2. A , SDS-PAGE analysis of purified proteins used in this study. GST alone, GST-Nox2-C, the wild-type p67 phox (1–212)-Rac1 (Q61L) chimera, and a mutant p67

    Article Snippet: For estimation of protein levels of FLAG-p47phox , Myc-p67phox , Myc-Rac1, and p22phox , proteins in cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-FLAG monoclonal antibody (Sigma-Aldrich), an anti-Myc monoclonal antibody (Roche Applied Science), and an anti-p22phox polyclonal antibody (Santa Cruz Biotechnology), respectively.

    Techniques: SDS Page, Purification, Mutagenesis

    Role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with Rac. A , SDS-PAGE analysis of purified GST fusion proteins that were used in a GST pull-down assay for interaction with Rac. GST alone and GST fusion protein of p67 phox -(1–301) (the wild-type ( wt ) or a mutant protein carrying the V204A, Y198A, L199A, Y198A/V204A, L199A/V204A, or R102E substitution) was subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue. The positions for marker proteins are indicated in kilodaltons. B , role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with Rac. GST-p67 phox proteins (the wild-type and indicated mutant proteins) were incubated with Rac1 (Q61L) and pulled down with glutathione-Sepharose 4B beads. The precipitated proteins were analyzed by immunoblot with the anti-Rac antibody, as described under “Experimental Procedures.” The data are representative of results from four independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase *

    doi: 10.1074/jbc.M110.161166

    Figure Lengend Snippet: Role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with Rac. A , SDS-PAGE analysis of purified GST fusion proteins that were used in a GST pull-down assay for interaction with Rac. GST alone and GST fusion protein of p67 phox -(1–301) (the wild-type ( wt ) or a mutant protein carrying the V204A, Y198A, L199A, Y198A/V204A, L199A/V204A, or R102E substitution) was subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue. The positions for marker proteins are indicated in kilodaltons. B , role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with Rac. GST-p67 phox proteins (the wild-type and indicated mutant proteins) were incubated with Rac1 (Q61L) and pulled down with glutathione-Sepharose 4B beads. The precipitated proteins were analyzed by immunoblot with the anti-Rac antibody, as described under “Experimental Procedures.” The data are representative of results from four independent experiments.

    Article Snippet: For estimation of protein levels of HA-p67phox , FLAG-p47phox , Myc-Rac1, and p22phox , proteins in cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-HA monoclonal antibody (Roche Applied Science), an anti-FLAG monoclonal antibody (Sigma-Aldrich), an anti-Myc monoclonal antibody (Roche Applied Science), and anti-p22phox polyclonal antibody (Santa Cruz Biotechnology), respectively.

    Techniques: SDS Page, Purification, Pull Down Assay, Mutagenesis, Staining, Marker, Incubation

    Role for the Nox activation region of p67 phox (amino acids 190–210) in gp91 phox /Nox2 activation. A , role for a region C-terminal to the TPR domain of p67 phox in a whole cell activation system of the phagocyte NADPH oxidase gp91 phox /Nox2. HA-tagged p67 phox (the wild-type ( wt ) or indicated mutant protein), FLAG-p47 phox , gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. B , role for Tyr-198, Leu-199, and Val-204 in activation of gp91 phox /Nox2. HA-tagged p67 phox carrying substitution for Tyr-198, Leu-199, or Val-204 as well as FLAG-p47 phox , gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. C , role for Tyr-198, Leu-199, and Val-204 in activation of gp91 phox /Nox2 in the presence of Rac1 (Q61L). HA-tagged p67 phox carrying the substitution for Tyr-198, Leu-199, or Val-204 as well as FLAG-p47 phox , Myc-Rac1 (Q61L), gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. Note that although the cDNA for Rac1 (Q61L) was transfected in C , neither wild-type nor mutant Rac1 was ectopically expressed in A and B . The protein levels of HA-p67 phox (the wild-type or indicated mutant protein), FLAG-p47 phox , Myc-Rac1 (Q61L), and p22 phox were analyzed by immunoblot with the anti-HA, anti-FLAG, anti-Myc, and anti-p22 phox antibodies, respectively, as described under “Experimental Procedures.” The transfected cells were incubated for 5 min at 37 °C and then stimulated with phorbol 12-myristate 13-acetate (200 ng/ml). The chemiluminescence change by the superoxide produced was continuously monitored with DIOGENES, as described under “Experimental Procedures.” Each graph represents the means ± S.D. of the chemiluminescence values integrated for 10 min, which were obtained from three independent transfections.

    Journal: The Journal of Biological Chemistry

    Article Title: A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase *

    doi: 10.1074/jbc.M110.161166

    Figure Lengend Snippet: Role for the Nox activation region of p67 phox (amino acids 190–210) in gp91 phox /Nox2 activation. A , role for a region C-terminal to the TPR domain of p67 phox in a whole cell activation system of the phagocyte NADPH oxidase gp91 phox /Nox2. HA-tagged p67 phox (the wild-type ( wt ) or indicated mutant protein), FLAG-p47 phox , gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. B , role for Tyr-198, Leu-199, and Val-204 in activation of gp91 phox /Nox2. HA-tagged p67 phox carrying substitution for Tyr-198, Leu-199, or Val-204 as well as FLAG-p47 phox , gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. C , role for Tyr-198, Leu-199, and Val-204 in activation of gp91 phox /Nox2 in the presence of Rac1 (Q61L). HA-tagged p67 phox carrying the substitution for Tyr-198, Leu-199, or Val-204 as well as FLAG-p47 phox , Myc-Rac1 (Q61L), gp91 phox /Nox2, and p22 phox were co-expressed in CHO cells. Note that although the cDNA for Rac1 (Q61L) was transfected in C , neither wild-type nor mutant Rac1 was ectopically expressed in A and B . The protein levels of HA-p67 phox (the wild-type or indicated mutant protein), FLAG-p47 phox , Myc-Rac1 (Q61L), and p22 phox were analyzed by immunoblot with the anti-HA, anti-FLAG, anti-Myc, and anti-p22 phox antibodies, respectively, as described under “Experimental Procedures.” The transfected cells were incubated for 5 min at 37 °C and then stimulated with phorbol 12-myristate 13-acetate (200 ng/ml). The chemiluminescence change by the superoxide produced was continuously monitored with DIOGENES, as described under “Experimental Procedures.” Each graph represents the means ± S.D. of the chemiluminescence values integrated for 10 min, which were obtained from three independent transfections.

    Article Snippet: For estimation of protein levels of HA-p67phox , FLAG-p47phox , Myc-Rac1, and p22phox , proteins in cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-HA monoclonal antibody (Roche Applied Science), an anti-FLAG monoclonal antibody (Sigma-Aldrich), an anti-Myc monoclonal antibody (Roche Applied Science), and anti-p22phox polyclonal antibody (Santa Cruz Biotechnology), respectively.

    Techniques: Activation Assay, Mutagenesis, Transfection, Incubation, Produced

    Role for the Nox activation region of p67 phox and the corresponding region of Noxa1 in Nox1 activation. A , activation of Nox1 by wild-type ( wt ) or mutant Noxa1 and Noxo1. HA-Noxa1 (the wild-type or indicated mutant protein), FLAG-Noxo1, Nox1, and p22 phox were co-expressed in CHO cells. B , activation of Nox1 by wild-type or mutant p67 phox and Noxo1 in the presence of Rac1 (Q61L). HA-p67 phox (the wild-type or indicated mutant protein), FLAG-Noxo1, Myc-Rac1 (Q61L), Nox1, and p22 phox were co-expressed in CHO cells. Protein levels of HA-p67 phox or HA-Noxa1, FLAG-Noxo1, Myc-Rac1 (Q61L), and p22 phox were analyzed by immunoblot with the anti-HA, anti-FLAG, anti-Myc, and anti-p22 phox antibodies, respectively, as described under “Experimental Procedures.” After preincubation for 5 min, the transfected cells were incubated at 37 °C with or without phorbol 12-myristate 13-acetate ( PMA , 200 ng/ml). Chemiluminescence change by superoxide produced was continuously monitored with DIOGENES, as described under “Experimental Procedures.” Each graph represents the means ± S.D. of the chemiluminescence values integrated for 10 min, which were obtained from three independent transfections.

    Journal: The Journal of Biological Chemistry

    Article Title: A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase *

    doi: 10.1074/jbc.M110.161166

    Figure Lengend Snippet: Role for the Nox activation region of p67 phox and the corresponding region of Noxa1 in Nox1 activation. A , activation of Nox1 by wild-type ( wt ) or mutant Noxa1 and Noxo1. HA-Noxa1 (the wild-type or indicated mutant protein), FLAG-Noxo1, Nox1, and p22 phox were co-expressed in CHO cells. B , activation of Nox1 by wild-type or mutant p67 phox and Noxo1 in the presence of Rac1 (Q61L). HA-p67 phox (the wild-type or indicated mutant protein), FLAG-Noxo1, Myc-Rac1 (Q61L), Nox1, and p22 phox were co-expressed in CHO cells. Protein levels of HA-p67 phox or HA-Noxa1, FLAG-Noxo1, Myc-Rac1 (Q61L), and p22 phox were analyzed by immunoblot with the anti-HA, anti-FLAG, anti-Myc, and anti-p22 phox antibodies, respectively, as described under “Experimental Procedures.” After preincubation for 5 min, the transfected cells were incubated at 37 °C with or without phorbol 12-myristate 13-acetate ( PMA , 200 ng/ml). Chemiluminescence change by superoxide produced was continuously monitored with DIOGENES, as described under “Experimental Procedures.” Each graph represents the means ± S.D. of the chemiluminescence values integrated for 10 min, which were obtained from three independent transfections.

    Article Snippet: For estimation of protein levels of HA-p67phox , FLAG-p47phox , Myc-Rac1, and p22phox , proteins in cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-HA monoclonal antibody (Roche Applied Science), an anti-FLAG monoclonal antibody (Sigma-Aldrich), an anti-Myc monoclonal antibody (Roche Applied Science), and anti-p22phox polyclonal antibody (Santa Cruz Biotechnology), respectively.

    Techniques: Activation Assay, Mutagenesis, Transfection, Incubation, Produced

    Role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with gp91 phox . A , SDS-PAGE analysis of purified, GST-tagged, or FLAG-tagged proteins that were used for interaction of p67 phox with gp91 phox . GST alone, GST-fused protein of gp91 phox -C, FLAG-tagged p67 phox (the wild-type ( wt ) or a mutant protein carrying the V204A, Y198A, L199A, Y198A/V204A, or L199A/V204A substitution), and Rac1 (Q61L) without a tag were subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue. The positions for marker proteins are indicated in kilodaltons. B , role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with gp91 phox . GST-gp91 phox -C was incubated with FLAG-p67 phox proteins (the wild-type and indicated mutant proteins) in the presence of Rac1 (Q61L) and pulled down with glutathione-Sepharose 4B beads. The precipitated proteins were analyzed by immunoblot with the anti-FLAG antibody, as described under “Experimental Procedures.” The data are representative of results from four independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase *

    doi: 10.1074/jbc.M110.161166

    Figure Lengend Snippet: Role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with gp91 phox . A , SDS-PAGE analysis of purified, GST-tagged, or FLAG-tagged proteins that were used for interaction of p67 phox with gp91 phox . GST alone, GST-fused protein of gp91 phox -C, FLAG-tagged p67 phox (the wild-type ( wt ) or a mutant protein carrying the V204A, Y198A, L199A, Y198A/V204A, or L199A/V204A substitution), and Rac1 (Q61L) without a tag were subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue. The positions for marker proteins are indicated in kilodaltons. B , role for Tyr-198, Leu-199, and Val-204 of p67 phox in interaction with gp91 phox . GST-gp91 phox -C was incubated with FLAG-p67 phox proteins (the wild-type and indicated mutant proteins) in the presence of Rac1 (Q61L) and pulled down with glutathione-Sepharose 4B beads. The precipitated proteins were analyzed by immunoblot with the anti-FLAG antibody, as described under “Experimental Procedures.” The data are representative of results from four independent experiments.

    Article Snippet: For estimation of protein levels of HA-p67phox , FLAG-p47phox , Myc-Rac1, and p22phox , proteins in cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore), and probed with an anti-HA monoclonal antibody (Roche Applied Science), an anti-FLAG monoclonal antibody (Sigma-Aldrich), an anti-Myc monoclonal antibody (Roche Applied Science), and anti-p22phox polyclonal antibody (Santa Cruz Biotechnology), respectively.

    Techniques: SDS Page, Purification, Mutagenesis, Staining, Marker, Incubation

    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of GTP (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of Rac1 GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value

    Journal: Oncotarget

    Article Title: CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

    doi: 10.18632/oncotarget.8638

    Figure Lengend Snippet: CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of GTP (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of Rac1 GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value

    Article Snippet: GTP-RAS and GTP-RAC1 pull-down assay The pull-down of GTP-bound RAS and RAC1 was performed by the use of active RAS and RAC1 pull-down and detection kits (Millipore) respectively according to the manufacturer's instructions.

    Techniques: Migration, Western Blot, Inhibition, Activity Assay, Incubation, Positive Control, Negative Control, Binding Assay, MTS Assay

    ARV-activated Rac1 through activation of p38 MAPK and Src. DF-1 cells were grown in 6-cm 2 cell culture dishes to 75% confluence, and then the cells were cultured in serum-free media overnight. Pulldown assay for GTP-Rac1 was performed. A , DF-1 cells were

    Journal: The Journal of Biological Chemistry

    Article Title: Cell Entry of Avian Reovirus Follows a Caveolin-1-mediated and Dynamin-2-dependent Endocytic Pathway That Requires Activation of p38 Mitogen-activated Protein Kinase (MAPK) and Src Signaling Pathways as Well as Microtubules and Small GTPase Rab5 Protein *

    doi: 10.1074/jbc.M111.257154

    Figure Lengend Snippet: ARV-activated Rac1 through activation of p38 MAPK and Src. DF-1 cells were grown in 6-cm 2 cell culture dishes to 75% confluence, and then the cells were cultured in serum-free media overnight. Pulldown assay for GTP-Rac1 was performed. A , DF-1 cells were

    Article Snippet: GTP-bound Rac1 was isolated from DF-1 cell lysates using a Rac1 activation assay kit (Millipore; San Diego) according to the manufacturer's protocol.

    Techniques: Activation Assay, Cell Culture