recombinant his-rac1 protein Search Results


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  • 99
    Thermo Fisher recombinant human rac1 gst
    Phosphorylation and activation of small GTPase <t>RAC1/Cdc42</t> proteins are reduced in Lrrk1-deficient osteoclasts. A : Western blot analyses show reduced phosphorylation of RAC1/Cdc42 at Ser 71 . B : Pull-down assays show reduced RAC1 binding to p21 protein-activated kinase binding domain (PAK-BD). C : RAC1 interacts with hLrrk1 in Raw 264.7 cells in response to RANKL treatment in a time-dependent manner, detected by coimmunoprecipitation. D : Western blot analyses show comparable expression of Akt phosphorylation at Ser 473 in WT and Lrrk1 KO osteoclasts. E : <t>RAC1-GST</t> was phosphorylated by recombinant mLrrk1 expressed in vitro. F : Western blot analyses show reduced phosphorylation of PAK1 at Ser 144 and Thr 423 in Lrrk1 KO osteoclasts compared with WT cells.
    Recombinant Human Rac1 Gst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gdp
    RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), <t>p.A57G</t> and p.F82L proteins (0.5 μM each) were loaded with <t>GDP</t> or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.
    Gdp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Cytoskeleton Inc his tagged rac1
    BART inhibits the translocation of <t>Rac1</t> to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.
    His Tagged Rac1, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 81/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare recombinant protein
    BART inhibits the translocation of <t>Rac1</t> to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.
    Recombinant Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 8288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Upstate Biotechnology Inc recombinant
    BART inhibits the translocation of <t>Rac1</t> to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.
    Recombinant, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Upstate Biotechnology Inc recombinant src pp60c src catalogue
    BART inhibits the translocation of <t>Rac1</t> to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.
    Recombinant Src Pp60c Src Catalogue, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore gst fused recombinant full length human aurora a
    Arpc1b: a new <t>Aurora</t> A substrate. (A) Kinase assay for <t>GST-Arpc1b</t> and GST-Arpc1b T21A proteins in presence of Aurora A. Aurora A in the middle panel indicates comparable amounts of protein loading. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for the study. Asterisk denotes the GST-fused protein of interest. (B) In vivo phosphorylation of T7-tagged Arpc1b in the Pak1 −/− MEFs treated with control or Arpc1b siRNA. (Top to bottom): [ 32 P]T7-Arpc1b, T7-Arpc1b, Aurora A, and vinculin. (C) Western blot showing extent of Arpc2 immunoprecipitated with Arpc1b from Pak1 +/+ or Pak1 −/− cells after Aurora A knockdown (top panel), effective Arpc1b pull-down (second panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (D) Western blot showing extent of Arpc1b immunoprecipitated with Arpc2 from the Pak1 −/− cells after Aurora A knockdown (second panel), effective Arpc2 pull-down (top panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (E) Pak1 −/− cells (synchronized in G1-S phase) transfected with either control or Arpc1b siRNAs were scored for the percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F) Proposed model explaining the role for threonine 21 phosphorylation on Arpc1b in Aurora A activation leading to centrosome amplification. WB, Western blot; IP, immunoprecipitation; kD, kilodaltons.
    Gst Fused Recombinant Full Length Human Aurora A, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Fisher Scientific recombinant human egf
    Arpc1b: a new <t>Aurora</t> A substrate. (A) Kinase assay for <t>GST-Arpc1b</t> and GST-Arpc1b T21A proteins in presence of Aurora A. Aurora A in the middle panel indicates comparable amounts of protein loading. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for the study. Asterisk denotes the GST-fused protein of interest. (B) In vivo phosphorylation of T7-tagged Arpc1b in the Pak1 −/− MEFs treated with control or Arpc1b siRNA. (Top to bottom): [ 32 P]T7-Arpc1b, T7-Arpc1b, Aurora A, and vinculin. (C) Western blot showing extent of Arpc2 immunoprecipitated with Arpc1b from Pak1 +/+ or Pak1 −/− cells after Aurora A knockdown (top panel), effective Arpc1b pull-down (second panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (D) Western blot showing extent of Arpc1b immunoprecipitated with Arpc2 from the Pak1 −/− cells after Aurora A knockdown (second panel), effective Arpc2 pull-down (top panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (E) Pak1 −/− cells (synchronized in G1-S phase) transfected with either control or Arpc1b siRNAs were scored for the percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F) Proposed model explaining the role for threonine 21 phosphorylation on Arpc1b in Aurora A activation leading to centrosome amplification. WB, Western blot; IP, immunoprecipitation; kD, kilodaltons.
    Recombinant Human Egf, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Cytoskeleton Inc p50 rhogap protein
    BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human <t>p50</t> <t>RhoGAP</t> protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P
    P50 Rhogap Protein, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare protein a sepharose beads
    BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human <t>p50</t> <t>RhoGAP</t> protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P
    Protein A Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 8304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti rac1
    Role of <t>Rac1</t> in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active
    Anti Rac1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher ni nta
    Role of <t>Rac1</t> in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active
    Ni Nta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt
    Role of <t>Rac1</t> in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active
    Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology angiopoietin 1 2
    Role of <t>Rac1</t> in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active
    Angiopoietin 1 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti β catenin
    sFRP-1 induction of EC spreading is independent of <t>β-catenin</t> and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
    Anti β Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho ser9 gsk 3β
    sFRP-1 induction of EC spreading is independent of β-catenin and requires <t>GSK-3β</t> activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
    Anti Phospho Ser9 Gsk 3β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa anti his
    sFRP-1 induction of EC spreading is independent of β-catenin and requires <t>GSK-3β</t> activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
    Anti His, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti α tubulin
    sFRP-1 induction of EC spreading is independent of β-catenin and requires <t>GSK-3β</t> activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated
    Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore non hydrolyzable gtpγs
    RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable <t>GTPγS</t> as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.
    Non Hydrolyzable Gtpγs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti akt
    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and <t>PI3K-Akt</t> signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of <t>Daple-Gαi3</t> and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 13710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytoskeleton Inc immunoblotting analysis
    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and <t>PI3K-Akt</t> signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of <t>Daple-Gαi3</t> and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
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    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and <t>PI3K-Akt</t> signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of <t>Daple-Gαi3</t> and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
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    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and <t>PI3K-Akt</t> signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of <t>Daple-Gαi3</t> and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
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    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and <t>PI3K-Akt</t> signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of <t>Daple-Gαi3</t> and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007
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    Cleavage of endogenous Rac GTPases by recombinant caspase 3 in vitro. Whole-cell extracts from healthy JLP-119 cells containing endogenous Rac GTPases were incubated for different times at 37°C with purified recombinant caspase 3 or 8 at final concentrations of 5 ng/μl in a caspase reaction buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% (wt/vol) sucrose, and 10 mM <t>dithiothreitol.</t> Proteins were examined by Western blot immunoassay with the indicated antibodies.
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    Image Search Results


    Phosphorylation and activation of small GTPase RAC1/Cdc42 proteins are reduced in Lrrk1-deficient osteoclasts. A : Western blot analyses show reduced phosphorylation of RAC1/Cdc42 at Ser 71 . B : Pull-down assays show reduced RAC1 binding to p21 protein-activated kinase binding domain (PAK-BD). C : RAC1 interacts with hLrrk1 in Raw 264.7 cells in response to RANKL treatment in a time-dependent manner, detected by coimmunoprecipitation. D : Western blot analyses show comparable expression of Akt phosphorylation at Ser 473 in WT and Lrrk1 KO osteoclasts. E : RAC1-GST was phosphorylated by recombinant mLrrk1 expressed in vitro. F : Western blot analyses show reduced phosphorylation of PAK1 at Ser 144 and Thr 423 in Lrrk1 KO osteoclasts compared with WT cells.

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation

    doi: 10.1152/ajpendo.00189.2016

    Figure Lengend Snippet: Phosphorylation and activation of small GTPase RAC1/Cdc42 proteins are reduced in Lrrk1-deficient osteoclasts. A : Western blot analyses show reduced phosphorylation of RAC1/Cdc42 at Ser 71 . B : Pull-down assays show reduced RAC1 binding to p21 protein-activated kinase binding domain (PAK-BD). C : RAC1 interacts with hLrrk1 in Raw 264.7 cells in response to RANKL treatment in a time-dependent manner, detected by coimmunoprecipitation. D : Western blot analyses show comparable expression of Akt phosphorylation at Ser 473 in WT and Lrrk1 KO osteoclasts. E : RAC1-GST was phosphorylated by recombinant mLrrk1 expressed in vitro. F : Western blot analyses show reduced phosphorylation of PAK1 at Ser 144 and Thr 423 in Lrrk1 KO osteoclasts compared with WT cells.

    Article Snippet: Recombinant human RAC1-GST, Ni-NTA His tag purification kit, and anti-mouse IgG magnetic beads were obtained from Life Technologies (Carlsbad, CA).

    Techniques: Activation Assay, Western Blot, Binding Assay, Expressing, Recombinant, In Vitro

    RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.

    Journal: PLoS Genetics

    Article Title: RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1

    doi: 10.1371/journal.pgen.1007370

    Figure Lengend Snippet: RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.

    Article Snippet: Depending on the experimental design, recombinant His-RIT1WT/A57G/F82L , GST-CDC42 and GST-RAC1 were coupled to GDP (G7127, Sigma-Aldrich) or non-hydrolyzable GTPγS (G8634, Sigma-Aldrich) for 30 min by incubation with 20 mM Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 2.5 mM EDTA and 1 mM of GDP or GTPγS at room temperature.

    Techniques: Recombinant, Incubation

    Expression of RIT1 mutants enhances phosphorylation of ERK1/2. (A) Schematic representation of RIT1 with selected NS-associated amino acid substitutions. RIT1 isoform 2 (protein RefSeq NP_008843.1) comprises 219 amino acids and has five conserved GDP/GTP binding motifs (G1 to G5, light blue). Motifs representing the P-loop and switch I and II regions are shown in dark blue. The P-loop binds γ-phosphate of GTP and GDP, the switch regions are critical for GDP/GTP binding and for interaction with upstream and downstream partners. RIT1 amino acid substitutions identified in patients with NS and selected for functional studies in this work are given in the one-letter code above the scheme. (B) HEK293T cells were transfected with empty vector (EV) or constructs expressing HA-RIT wildtype (WT), HA-RIT1 p.A57G, p.F82L, or p.G95A as indicated. Cells were serum-starved (0.1% serum) and subsequently stimulated with 20% serum for 5, 15, or 30 min or left untreated (0 min). Cell extracts were analyzed by immunoblotting using anti-phospho-ERK1/2 (pERK1/2) and anti-ERK1/2 (ERK1/2) antibodies. Expression of HA-tagged RIT1 protein variants was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was used to control for equal loading. Data shown are representative of three independent experiments. Autoradiographic signals were quantified by scanning densitometry. Levels of phosphorylated ERK1/2 were normalized relative to amounts of total ERK1/2. To conserve the relative variance of the samples, values for RIT1 wildtype and mutants were divided by the mean of the wildtype samples [ 79 ]. Graphs show phosphorylation levels upon serum starvation (0 min) and after 5, 15, and 30 min serum stimulation in cells expressing RIT1 wildtype (WT), RIT1 p.A57G, p.F82L or p.G95A (arbitrary units). The mean of three independent experiments ± SD is given. Unpaired t -tests were used to determine statistical significance (*, P

    Journal: PLoS Genetics

    Article Title: RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1

    doi: 10.1371/journal.pgen.1007370

    Figure Lengend Snippet: Expression of RIT1 mutants enhances phosphorylation of ERK1/2. (A) Schematic representation of RIT1 with selected NS-associated amino acid substitutions. RIT1 isoform 2 (protein RefSeq NP_008843.1) comprises 219 amino acids and has five conserved GDP/GTP binding motifs (G1 to G5, light blue). Motifs representing the P-loop and switch I and II regions are shown in dark blue. The P-loop binds γ-phosphate of GTP and GDP, the switch regions are critical for GDP/GTP binding and for interaction with upstream and downstream partners. RIT1 amino acid substitutions identified in patients with NS and selected for functional studies in this work are given in the one-letter code above the scheme. (B) HEK293T cells were transfected with empty vector (EV) or constructs expressing HA-RIT wildtype (WT), HA-RIT1 p.A57G, p.F82L, or p.G95A as indicated. Cells were serum-starved (0.1% serum) and subsequently stimulated with 20% serum for 5, 15, or 30 min or left untreated (0 min). Cell extracts were analyzed by immunoblotting using anti-phospho-ERK1/2 (pERK1/2) and anti-ERK1/2 (ERK1/2) antibodies. Expression of HA-tagged RIT1 protein variants was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was used to control for equal loading. Data shown are representative of three independent experiments. Autoradiographic signals were quantified by scanning densitometry. Levels of phosphorylated ERK1/2 were normalized relative to amounts of total ERK1/2. To conserve the relative variance of the samples, values for RIT1 wildtype and mutants were divided by the mean of the wildtype samples [ 79 ]. Graphs show phosphorylation levels upon serum starvation (0 min) and after 5, 15, and 30 min serum stimulation in cells expressing RIT1 wildtype (WT), RIT1 p.A57G, p.F82L or p.G95A (arbitrary units). The mean of three independent experiments ± SD is given. Unpaired t -tests were used to determine statistical significance (*, P

    Article Snippet: Depending on the experimental design, recombinant His-RIT1WT/A57G/F82L , GST-CDC42 and GST-RAC1 were coupled to GDP (G7127, Sigma-Aldrich) or non-hydrolyzable GTPγS (G8634, Sigma-Aldrich) for 30 min by incubation with 20 mM Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 2.5 mM EDTA and 1 mM of GDP or GTPγS at room temperature.

    Techniques: Expressing, Binding Assay, Functional Assay, Transfection, Plasmid Preparation, Construct

    BART inhibits the translocation of Rac1 to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART inhibits the translocation of Rac1 to the plasma membrane. (A) The mock control vector or myc -tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti- myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti- myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µ m.

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Translocation Assay, Plasmid Preparation, Construct, Transfection, Incubation, Western Blot, Staining

    BART binds to Rac1 at the leading edges of migrating cells. (A) Immunoprecipitation of endogenous BART or Rac1 from S2-013 cells. Anti-BART or anti-Rac1 immunoprecipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Rabbit IgG and mouse IgG monoclonal antibodies were used as isotype controls for anti-BART and anti-Rac1 antibodies, respectively. Data are representative of three independent experiments. (B) Immunocytochemical staining of S2-013 cells using anti-BART (green) and anti-Rac1 (red) antibodies. Arrow indicates colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bar, 10 µ m. (C) Confluent S2-013 cells were wounded. After 4 hours, the cells were immunostained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 at the leading edge; blue, DAPI staining. Bar, 10 µ m.

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART binds to Rac1 at the leading edges of migrating cells. (A) Immunoprecipitation of endogenous BART or Rac1 from S2-013 cells. Anti-BART or anti-Rac1 immunoprecipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Rabbit IgG and mouse IgG monoclonal antibodies were used as isotype controls for anti-BART and anti-Rac1 antibodies, respectively. Data are representative of three independent experiments. (B) Immunocytochemical staining of S2-013 cells using anti-BART (green) and anti-Rac1 (red) antibodies. Arrow indicates colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bar, 10 µ m. (C) Confluent S2-013 cells were wounded. After 4 hours, the cells were immunostained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 at the leading edge; blue, DAPI staining. Bar, 10 µ m.

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Immunoprecipitation, Western Blot, Staining

    BART binds to an active form of Rac1. (A) GST-tagged Rac1 was incubated with GDP or GTPγS and was then used in pull-down experiments with the recombinant BART protein. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) GST-tagged Rac1, a dominant-negative mutated Rac1 form, Rac1N17, or PAK-CRIB was incubated with S2-013 cell lysates, followed by GST-pull-down assays. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (C) Densitometric analysis of the results of B. The level of BART in the precipitates was assessed after normalizing BART signals to Rac1 signals. (D) S2-013 cells were pretreated with or without the Rac1 inhibitor (NSC23766) and were immunocytochemically stained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bars, 10 µ m.

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART binds to an active form of Rac1. (A) GST-tagged Rac1 was incubated with GDP or GTPγS and was then used in pull-down experiments with the recombinant BART protein. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) GST-tagged Rac1, a dominant-negative mutated Rac1 form, Rac1N17, or PAK-CRIB was incubated with S2-013 cell lysates, followed by GST-pull-down assays. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (C) Densitometric analysis of the results of B. The level of BART in the precipitates was assessed after normalizing BART signals to Rac1 signals. (D) S2-013 cells were pretreated with or without the Rac1 inhibitor (NSC23766) and were immunocytochemically stained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bars, 10 µ m.

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Incubation, Recombinant, Western Blot, Dominant Negative Mutation, Staining

    BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human p50 RhoGAP protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human p50 RhoGAP protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Activity Assay, Construct, Transfection, Western Blot, In Vitro, Recombinant, Negative Control, Generated

    BART decreases the activity of Rac1 resulting in inhibition of PDAC cell motility and invasion. (A) The amount of active, GTP-loaded Rac1 in S2-013 and PANC-1 cells that were mock transfected (Neo-1) or transfected with control, scrambled (Scr-1), or BART (siBART-1 and 2) siRNA was determined using a GST-PAK-CRIB pull-down assay. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. (B) Two S2-013 clones transfected with siRNA for BART were pretreated with or without the Rac1 inhibitor (NSC23766), and the amount of active GTP-loaded Rac1 and Cdc42 and RhoA was analyzed with GST pull-down assays using GST-PAK-CRIB and GST-Rhotekin, respectively. Precipitates were examined by Western blot analysis using anti-Rac1, anti-Cdc42, and anti-RhoA antibodies. Total levels of Rac1, Cdc42, and RhoA protein were used to normalize the data. Data are representative of three independent experiments. (C) Confluent control and BART RNAi S2-013 cells treated as in B were wounded. The number of cells that migrated into an initially cell-free scratch was counted. Cells in four defined areas per group per experiment were quantified. Data are representative of three independent experiments. Bars, SD; columns, mean. * P

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART decreases the activity of Rac1 resulting in inhibition of PDAC cell motility and invasion. (A) The amount of active, GTP-loaded Rac1 in S2-013 and PANC-1 cells that were mock transfected (Neo-1) or transfected with control, scrambled (Scr-1), or BART (siBART-1 and 2) siRNA was determined using a GST-PAK-CRIB pull-down assay. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. (B) Two S2-013 clones transfected with siRNA for BART were pretreated with or without the Rac1 inhibitor (NSC23766), and the amount of active GTP-loaded Rac1 and Cdc42 and RhoA was analyzed with GST pull-down assays using GST-PAK-CRIB and GST-Rhotekin, respectively. Precipitates were examined by Western blot analysis using anti-Rac1, anti-Cdc42, and anti-RhoA antibodies. Total levels of Rac1, Cdc42, and RhoA protein were used to normalize the data. Data are representative of three independent experiments. (C) Confluent control and BART RNAi S2-013 cells treated as in B were wounded. The number of cells that migrated into an initially cell-free scratch was counted. Cells in four defined areas per group per experiment were quantified. Data are representative of three independent experiments. Bars, SD; columns, mean. * P

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Activity Assay, Inhibition, Transfection, Pull Down Assay, Western Blot, Clone Assay

    Arpc1b: a new Aurora A substrate. (A) Kinase assay for GST-Arpc1b and GST-Arpc1b T21A proteins in presence of Aurora A. Aurora A in the middle panel indicates comparable amounts of protein loading. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for the study. Asterisk denotes the GST-fused protein of interest. (B) In vivo phosphorylation of T7-tagged Arpc1b in the Pak1 −/− MEFs treated with control or Arpc1b siRNA. (Top to bottom): [ 32 P]T7-Arpc1b, T7-Arpc1b, Aurora A, and vinculin. (C) Western blot showing extent of Arpc2 immunoprecipitated with Arpc1b from Pak1 +/+ or Pak1 −/− cells after Aurora A knockdown (top panel), effective Arpc1b pull-down (second panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (D) Western blot showing extent of Arpc1b immunoprecipitated with Arpc2 from the Pak1 −/− cells after Aurora A knockdown (second panel), effective Arpc2 pull-down (top panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (E) Pak1 −/− cells (synchronized in G1-S phase) transfected with either control or Arpc1b siRNAs were scored for the percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F) Proposed model explaining the role for threonine 21 phosphorylation on Arpc1b in Aurora A activation leading to centrosome amplification. WB, Western blot; IP, immunoprecipitation; kD, kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

    doi: 10.1083/jcb.200908050

    Figure Lengend Snippet: Arpc1b: a new Aurora A substrate. (A) Kinase assay for GST-Arpc1b and GST-Arpc1b T21A proteins in presence of Aurora A. Aurora A in the middle panel indicates comparable amounts of protein loading. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for the study. Asterisk denotes the GST-fused protein of interest. (B) In vivo phosphorylation of T7-tagged Arpc1b in the Pak1 −/− MEFs treated with control or Arpc1b siRNA. (Top to bottom): [ 32 P]T7-Arpc1b, T7-Arpc1b, Aurora A, and vinculin. (C) Western blot showing extent of Arpc2 immunoprecipitated with Arpc1b from Pak1 +/+ or Pak1 −/− cells after Aurora A knockdown (top panel), effective Arpc1b pull-down (second panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (D) Western blot showing extent of Arpc1b immunoprecipitated with Arpc2 from the Pak1 −/− cells after Aurora A knockdown (second panel), effective Arpc2 pull-down (top panel), and effective knockdown of Aurora A (third panel). Vinculin was used as an internal control. (E) Pak1 −/− cells (synchronized in G1-S phase) transfected with either control or Arpc1b siRNAs were scored for the percentage of cells in G2-M phase at the indicated time points after release from G1-S arrest using FACS analysis; n = 3. (F) Proposed model explaining the role for threonine 21 phosphorylation on Arpc1b in Aurora A activation leading to centrosome amplification. WB, Western blot; IP, immunoprecipitation; kD, kilodaltons.

    Article Snippet: Anderson Cancer Center, Houston, TX). (His)6-tagged or GST-fused recombinant full-length human Aurora A was purchased from Millipore and Cell Signaling Technology, respectively.

    Techniques: Kinase Assay, Staining, In Vivo, Western Blot, Immunoprecipitation, Transfection, FACS, Activation Assay, Amplification

    Arpc1b localizes to the centrosome. (A) Synchronized ZR-75 cells were released for 6 h and analyzed for endogenous Arpc1b colocalization with centrosomal proteins Aurora A and γ-tubulin. Aurora A and γ-tubulin (green); endogenous Arpc1b (red); DNA (blue). Circle marks centrosome location. Bar, 5 µm. (B) Equal amount of cell extract was immunoprecipitated with control IgG or γ-tubulin antibody from the synchronized cells (top panel). The same blot was probed for Arpc1b (second last panel) and Arp3 (last panel). The blot was stripped and probed for Aurora A (second panel). (C) Equal amount of cell extract was immunoprecipitated with control IgG or Aurora A antibody (top panel). The same blot was probed for γ-tubulin (second panel) and Arp3 (last panel). Input, cell lysate used as positive control. (D) Xenopus egg extract was treated with either GST (control) or GST-WASP-CA (Arp3 depletion) and used for IP experiments. The Western blot shows Arp3 depletion in GST-WASP-CA–treated egg extract (top panel) and the GST-Aprc1b (second panel) used for the study. Aurora A was immunoprecipitated from the treated egg extract and incubated with GST-Arpc1b protein. The Western blot shows immunoprecipitated Aurora A and GST-Arpc1b or endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. (E) Aurora A was immunoprecipitated from the treated egg extract. Western blot shows immunoprecipitated Aurora A and endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. kD, kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

    doi: 10.1083/jcb.200908050

    Figure Lengend Snippet: Arpc1b localizes to the centrosome. (A) Synchronized ZR-75 cells were released for 6 h and analyzed for endogenous Arpc1b colocalization with centrosomal proteins Aurora A and γ-tubulin. Aurora A and γ-tubulin (green); endogenous Arpc1b (red); DNA (blue). Circle marks centrosome location. Bar, 5 µm. (B) Equal amount of cell extract was immunoprecipitated with control IgG or γ-tubulin antibody from the synchronized cells (top panel). The same blot was probed for Arpc1b (second last panel) and Arp3 (last panel). The blot was stripped and probed for Aurora A (second panel). (C) Equal amount of cell extract was immunoprecipitated with control IgG or Aurora A antibody (top panel). The same blot was probed for γ-tubulin (second panel) and Arp3 (last panel). Input, cell lysate used as positive control. (D) Xenopus egg extract was treated with either GST (control) or GST-WASP-CA (Arp3 depletion) and used for IP experiments. The Western blot shows Arp3 depletion in GST-WASP-CA–treated egg extract (top panel) and the GST-Aprc1b (second panel) used for the study. Aurora A was immunoprecipitated from the treated egg extract and incubated with GST-Arpc1b protein. The Western blot shows immunoprecipitated Aurora A and GST-Arpc1b or endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. (E) Aurora A was immunoprecipitated from the treated egg extract. Western blot shows immunoprecipitated Aurora A and endogenous Arpc1b in complex with Aurora A in control and Arp3-depleted egg extract. kD, kilodaltons.

    Article Snippet: Anderson Cancer Center, Houston, TX). (His)6-tagged or GST-fused recombinant full-length human Aurora A was purchased from Millipore and Cell Signaling Technology, respectively.

    Techniques: Immunoprecipitation, Positive Control, Western Blot, Incubation

    Arpc1b stimulates activity of Aurora A. (A) GST alone or GST-Arpc1b was used in an in vitro kinase assay with GST-Aurora A or GST-Aurora A KD protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 used for both reactions. Western blot shows equal amount of Aurora A was used in all the reactions. (B) GST, GST-Arpc1b, or GST-Arp3 was used in an in vitro kinase assay with recombinant Aurora A using Histone H3 as a substrate. Phosphorylated histone H3 was visualized by autoradiography (top panel). Ponceau-stained blot shows equal amount of GST proteins and histone H3 used for all the reactions. Asterisk denotes the GST-fused protein of interest. (C) Aurora A kinase activity was studied using histone H3 as a substrate in the presence or absence purified Arp2/3 complex. Ponceau-stained blot shows equal amount of histone H3 and the Western blot shows equal amount of Aurora A used in the reactions. (D) GST or GST-Arpc1b was incubated with phosphatase (PP1)-treated Aurora A for 20 min in presence of [ 32 P]γATP. Samples were analyzed for Aurora A phosphorylation (Thr-288, top panel) and total Aurora A (last panel). Fold change was expressed with respect to lane 1. (E) ZR-75 cells transfected with either control siRNA or Arpc1b siRNA were subjected to in vivo 32 P-labeling. Protein extracts were subjected to IP with an anti-Aurora A antibody, and phosphorylated Aurora A was visualized by autoradiography. Knockdown of Arpc1b was verified by Western blot with an anti-Arpc1b antibody (bottom panel). (F) Immunoblots showing levels of phospho-Aurora A (Thr288), phospho-plk1 (Thr 210), and phospho-histone H3 (Ser 10) in control and Arpc1b siRNA-treated ZR-75 cell lysates. kD, kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

    doi: 10.1083/jcb.200908050

    Figure Lengend Snippet: Arpc1b stimulates activity of Aurora A. (A) GST alone or GST-Arpc1b was used in an in vitro kinase assay with GST-Aurora A or GST-Aurora A KD protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 used for both reactions. Western blot shows equal amount of Aurora A was used in all the reactions. (B) GST, GST-Arpc1b, or GST-Arp3 was used in an in vitro kinase assay with recombinant Aurora A using Histone H3 as a substrate. Phosphorylated histone H3 was visualized by autoradiography (top panel). Ponceau-stained blot shows equal amount of GST proteins and histone H3 used for all the reactions. Asterisk denotes the GST-fused protein of interest. (C) Aurora A kinase activity was studied using histone H3 as a substrate in the presence or absence purified Arp2/3 complex. Ponceau-stained blot shows equal amount of histone H3 and the Western blot shows equal amount of Aurora A used in the reactions. (D) GST or GST-Arpc1b was incubated with phosphatase (PP1)-treated Aurora A for 20 min in presence of [ 32 P]γATP. Samples were analyzed for Aurora A phosphorylation (Thr-288, top panel) and total Aurora A (last panel). Fold change was expressed with respect to lane 1. (E) ZR-75 cells transfected with either control siRNA or Arpc1b siRNA were subjected to in vivo 32 P-labeling. Protein extracts were subjected to IP with an anti-Aurora A antibody, and phosphorylated Aurora A was visualized by autoradiography. Knockdown of Arpc1b was verified by Western blot with an anti-Arpc1b antibody (bottom panel). (F) Immunoblots showing levels of phospho-Aurora A (Thr288), phospho-plk1 (Thr 210), and phospho-histone H3 (Ser 10) in control and Arpc1b siRNA-treated ZR-75 cell lysates. kD, kilodaltons.

    Article Snippet: Anderson Cancer Center, Houston, TX). (His)6-tagged or GST-fused recombinant full-length human Aurora A was purchased from Millipore and Cell Signaling Technology, respectively.

    Techniques: Activity Assay, In Vitro, Kinase Assay, Autoradiography, Staining, Western Blot, Recombinant, Purification, Incubation, Transfection, In Vivo, Labeling

    Arpc1b interacts with Aurora kinase A. (A) ZR-75 cells synchronized by double-thymidine block and released for 6 h were analyzed for centrosome numbers. α-Tubulin (green) is used to indicate spindle morphology and pericentrin (red) is used to indicate centrosomes. DNA is in blue. N, number of centrosomes per cell. Quantitation for the centrosome numbers in the different stable cell lines generated in ZR-75 cells is shown below panel A. Bar, 20 µm. (B) ZR-75 cell lysates were incubated with either GST or GST-Arpc1b and the protein complex precipitated was subjected to Western blot analysis with the antibodies. Ponceau-stained blot shows the quality of GST proteins used for the study. (C) GST control or GST-Aurora A was incubated with purified Arp2/3 complex and the immunoprecipitates were subjected to Western blot analysis using an anti-Arpc1b or anti-Arpc2 antibody. (D) ZR-75 cell extracts were fractionated onto 3–30% sucrose gradients by velocity sedimentation, and then subjected to Western blot analysis with the indicated antibodies. (E) GST-tagged deletion mutants of human Aurora kinase A. Aurora A, 1–403 is the full-length protein. In vitro–translated 35 S-labeled Arpc1b was used to study binding of different GST-Aurora A deletion mutants. The extent of binding was estimated by measuring signal intensity. Ponceau S staining shows equal quantity of GST and GST-Aurora A deletions used in the reaction. Asterisk denotes the GST-fused protein of interest; black arrows indicate the interacting region of Aurora kinase A with Arpc1b. (F) Asynchronized cells or cells blocked with thymidine and released for various time points were used for immunoprecipitation (IP). Transfected T7-Arpc1b was immunoprecipitated using T7 antibody and the blot was probed for Aurora A. (G) Asynchronized cells or cells blocked with thymidine and released for various time points were subjected to the sequential IP/Western blot with the indicated antibodies. (H) Protein extract from ZR-75 cells released for 0 or 8 h after double-thymidine block were subjected to reciprocal immunoprecipitation assays with an anti-Aurora A or anti-Arpc1b antibody, followed by Western blot analysis with the indicated antibodies. kD, kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

    doi: 10.1083/jcb.200908050

    Figure Lengend Snippet: Arpc1b interacts with Aurora kinase A. (A) ZR-75 cells synchronized by double-thymidine block and released for 6 h were analyzed for centrosome numbers. α-Tubulin (green) is used to indicate spindle morphology and pericentrin (red) is used to indicate centrosomes. DNA is in blue. N, number of centrosomes per cell. Quantitation for the centrosome numbers in the different stable cell lines generated in ZR-75 cells is shown below panel A. Bar, 20 µm. (B) ZR-75 cell lysates were incubated with either GST or GST-Arpc1b and the protein complex precipitated was subjected to Western blot analysis with the antibodies. Ponceau-stained blot shows the quality of GST proteins used for the study. (C) GST control or GST-Aurora A was incubated with purified Arp2/3 complex and the immunoprecipitates were subjected to Western blot analysis using an anti-Arpc1b or anti-Arpc2 antibody. (D) ZR-75 cell extracts were fractionated onto 3–30% sucrose gradients by velocity sedimentation, and then subjected to Western blot analysis with the indicated antibodies. (E) GST-tagged deletion mutants of human Aurora kinase A. Aurora A, 1–403 is the full-length protein. In vitro–translated 35 S-labeled Arpc1b was used to study binding of different GST-Aurora A deletion mutants. The extent of binding was estimated by measuring signal intensity. Ponceau S staining shows equal quantity of GST and GST-Aurora A deletions used in the reaction. Asterisk denotes the GST-fused protein of interest; black arrows indicate the interacting region of Aurora kinase A with Arpc1b. (F) Asynchronized cells or cells blocked with thymidine and released for various time points were used for immunoprecipitation (IP). Transfected T7-Arpc1b was immunoprecipitated using T7 antibody and the blot was probed for Aurora A. (G) Asynchronized cells or cells blocked with thymidine and released for various time points were subjected to the sequential IP/Western blot with the indicated antibodies. (H) Protein extract from ZR-75 cells released for 0 or 8 h after double-thymidine block were subjected to reciprocal immunoprecipitation assays with an anti-Aurora A or anti-Arpc1b antibody, followed by Western blot analysis with the indicated antibodies. kD, kilodaltons.

    Article Snippet: Anderson Cancer Center, Houston, TX). (His)6-tagged or GST-fused recombinant full-length human Aurora A was purchased from Millipore and Cell Signaling Technology, respectively.

    Techniques: Blocking Assay, Quantitation Assay, Stable Transfection, Generated, Incubation, Western Blot, Staining, Purification, Sedimentation, In Vitro, Labeling, Binding Assay, Immunoprecipitation, Transfection

    Phosphorylation of Arpc1b on Thr 21 regulates its interaction with Aurora A. (A) In vitro–translated 35 S-labeled Aurora A was used to study its binding with GST, GST-Arpc1b, or GST-Arpc1b T21A. The extent of binding was estimated by measuring signal intensity. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for each reaction. (B) GST, GST-Arpc1b, and GST-Arpc1b T21A were used in an in vitro kinase assay with recombinant Aurora A protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 and GST-tagged proteins used for the study. Circle marks GST-fused protein of interest. (C) The 1C1 monoclonal antibody to Xenopus Aurora A (Eg2) was used to immunoprecipitate Eg2 from CSF egg extract supplemented with GST control, GST-TPX2 (1–126), GST-Arpc1b, or GST-Arpc1b T21A and the bound proteins were analyzed by Western blotting using anti-GST antibodies. Asterisk denotes the GST-fused protein of interest. Bar plot shows fold change quantitated from three experiments. (D) A phospho-specific antibody that recognizes phospho-Thr 295 of Eg2 was used to study the extent of activation of Aurora A (top panel). Bottom panel shows equal amount of Aurora A used in all the reactions. Bar plot shows fold change quantitated from three experiments. kD, kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A

    doi: 10.1083/jcb.200908050

    Figure Lengend Snippet: Phosphorylation of Arpc1b on Thr 21 regulates its interaction with Aurora A. (A) In vitro–translated 35 S-labeled Aurora A was used to study its binding with GST, GST-Arpc1b, or GST-Arpc1b T21A. The extent of binding was estimated by measuring signal intensity. Ponceau-stained blot shows equal quantity of GST-tagged proteins used for each reaction. (B) GST, GST-Arpc1b, and GST-Arpc1b T21A were used in an in vitro kinase assay with recombinant Aurora A protein. Phosphorylated histone H3 was visualized by autoradiography. Ponceau-stained blot shows equal amount of histone H3 and GST-tagged proteins used for the study. Circle marks GST-fused protein of interest. (C) The 1C1 monoclonal antibody to Xenopus Aurora A (Eg2) was used to immunoprecipitate Eg2 from CSF egg extract supplemented with GST control, GST-TPX2 (1–126), GST-Arpc1b, or GST-Arpc1b T21A and the bound proteins were analyzed by Western blotting using anti-GST antibodies. Asterisk denotes the GST-fused protein of interest. Bar plot shows fold change quantitated from three experiments. (D) A phospho-specific antibody that recognizes phospho-Thr 295 of Eg2 was used to study the extent of activation of Aurora A (top panel). Bottom panel shows equal amount of Aurora A used in all the reactions. Bar plot shows fold change quantitated from three experiments. kD, kilodaltons.

    Article Snippet: Anderson Cancer Center, Houston, TX). (His)6-tagged or GST-fused recombinant full-length human Aurora A was purchased from Millipore and Cell Signaling Technology, respectively.

    Techniques: In Vitro, Labeling, Binding Assay, Staining, Kinase Assay, Recombinant, Autoradiography, Western Blot, Activation Assay

    BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human p50 RhoGAP protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P

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

    Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

    doi:

    Figure Lengend Snippet: BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human p50 RhoGAP protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. * P

    Article Snippet: His-tagged Rac1 (5 µg; Cytoskeleton) was incubated with recombinant BART protein (0.5 µmol) and 200 µM GTP with or without the p50 RhoGAP protein (0.5 µmol; Cytoskeleton) at 37°C for 20 minutes in a reaction mixture (20 µl) containing 25 mM HEPES (pH 7.4), 100 mM NaCl, 2 mM MgCl2 , and 1 mM DTT.

    Techniques: Activity Assay, Construct, Transfection, Western Blot, In Vitro, Recombinant, Negative Control, Generated

    Role of Rac1 in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of Rac1 in sFRP-1 induced spreading on endothelial cells. sFRP-1 leads to an activation of Rac1, which is required for sFRP-1-induced EC spreading. a: Immunoprecipitation using a GST-human PAK1-PBD fusion protein that binds to GTP-bound Rac1 (active

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Immunoprecipitation

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Infection, Expressing

    RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.

    Journal: PLoS Genetics

    Article Title: RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1

    doi: 10.1371/journal.pgen.1007370

    Figure Lengend Snippet: RIT1 directly interacts with PAK1. Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1 GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1 GTPγS are indicated in μM. As control, His-RIT1 GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.

    Article Snippet: Depending on the experimental design, recombinant His-RIT1WT/A57G/F82L , GST-CDC42 and GST-RAC1 were coupled to GDP (G7127, Sigma-Aldrich) or non-hydrolyzable GTPγS (G8634, Sigma-Aldrich) for 30 min by incubation with 20 mM Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 2.5 mM EDTA and 1 mM of GDP or GTPγS at room temperature.

    Techniques: Recombinant, Incubation

    Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and PI3K-Akt signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of Daple-Gαi3 and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007

    Journal: eLife

    Article Title: Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

    doi: 10.7554/eLife.07091

    Figure Lengend Snippet: Daple's GBA motif triggers the release of ‘free’ Gβγ subunits, which in turn enhance Rac1 and PI3K-Akt signaling. ( A ) Daple's GBA motif and Gβγ subunits are predicted to dock onto an overlapping binding site on Gαi. Binding areas (in red) for Daple (left) or Gβγ (right) on Gαi (solid gray) were extracted from a homology-based model of Daple-Gαi3 and the crystal structure of the Gαi1·Gβγ complex (Protein Data Bank [PDB]: 1GG2), respectively. ( B , C ) Daple displaces Gβγ subunits from Gαi3 via its GBA motif. GST-Gαi3·Gβγ preformed complexes immobilized on glutathione beads were incubated with increasing concentrations of His-Daple-CT WT or F1675A (FA). Bound proteins were analyzed by IB ( B ) and Gβγ binding data fitted to a single-site competition curve ( C ). Mean ± S.E.M. of three independent experiments. ( D , E ) Activation of Rac1 is impaired in Daple-depleted HeLa cells. Control (shLuc) or two clones of Daple-depleted HeLa cell lines (sh Daple 1 and 2) (described in Figure 2—figure supplement 1A,B ) were incubated in 2% serum media ( D ) or starved and treated (+) or not (−) with Wnt5a (0.1 mg/ml) for 5 min ( E ) and analyzed for Rac1 activation by pulldown assays using GST-PBD. ( F ) Activation of Rac1 is impaired in cells expressing Daple-F1675A (FA) mutant compared to those expressing Daple-WT. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple-WT or FA were starved and stimulated with Wnt5a and analyzed for Rac1 activation as in E . ( G , H ) Daple's GBA motif is required for activation of PI3K-Akt signaling in HeLa cells, as determined by phosphorylation of Akt at S473. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT or F1675A (FA) were incubated in a 2% serum media ( G ) or in a 0.2% serum media overnight and treated (+) or not (−) with 0.1 mg/ml Wnt5a for 5 min ( H ) prior to lysis. Equal aliquots of whole-cell lysates were analyzed for Akt phosphorylation (pAkt S473) by IB. ( I , J ) Inhibition of Gβγ signaling impairs Daple-dependent activation of Rac1 and Akt. Daple-depleted (sh Daple 1) HeLa cells transiently transfected with myc-Daple WT were treated with DMSO, 10 µM of the Gβγ inhibitor gallein or its inactive analog fluorescein for 6 hr, as indicated, and analyzed for Rac1 ( I ) or Akt ( J ) activation by IB or pulldown assays, respectively. DOI: http://dx.doi.org/10.7554/eLife.07091.007

    Article Snippet: Rabbit anti-pan-Gβ (M-14), anti-Gαi3, anti-DVL, and anti-β-catenin were obtained from Santa Cruz Biotechnology (Dallas, TX); anti-Akt and phospho-Akt (S473) were obtained from Cell Signaling; anti-Rac1 was obtained from BD Transduction Laboratories (San Jose, CA).

    Techniques: Binding Assay, Incubation, Activation Assay, Clone Assay, Expressing, Mutagenesis, Transfection, Lysis, Inhibition

    Daple competes with Dvl for binding to FZD7R and inhibits the canonical β-caenin/TCF/LEF signaling pathway. ( A ) Equal aliquots of lysates from Cos7 cells expressing Dvl1 alone (lane 2), myc-Daple alone (lane 3), or coexpressing both (lanes 1, 4) were used as source of Daple and Dvl in GST pulldown assays with recombinant, immobilized GST or GST-FZD7-CT. Bound proteins were analyzed for Dvl1 and Daple by immunoblotting (IB). Binding of each protein was higher when expressed alone (lanes 2, 3) than when co-expressed (lane 4). ( B ) Dvl loses colocalization with FZD7R at the PM after Wnt5a stimulation. HEK293 cells expressing FZD7-CFP were grown on cover slips coated with Poly-D-Lysine, starved overnight, and treated with 0.1 mg/ml Wnt5a as in 4B. Cells were fixed and stained for endogenous Dvl (red) and analyzed by confocal microscopy. ( C , D ) Generation and characterization of DLD1 7TGP cell lines stably expressing Daple. ( C ) DLD1 7TGP cell lines stably expressing Daple-WT or FA were starved and stimulated analyzed for Daple expression and phosphorylation of Akt by immunoblotting (IB). ( D ) Images display representative fields from monolayers of DLD1 cells grown in 0.2% FBS by fluorescence microscopy. The intensity of eGFP signals denotes Wnt transcriptional activity. Inset shows immunoblots (IB) of equal aliquots of whole-cell lysates of DLD1-7TGP cells expressing control vector, Daple-WT, or Daple-FA. Compared to DLD1 cells expressing Daple-WT, those expressing Daple-FA also express higher levels of GFP protein, indicative of higher Wnt transcriptional activity. DOI: http://dx.doi.org/10.7554/eLife.07091.012

    Journal: eLife

    Article Title: Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

    doi: 10.7554/eLife.07091

    Figure Lengend Snippet: Daple competes with Dvl for binding to FZD7R and inhibits the canonical β-caenin/TCF/LEF signaling pathway. ( A ) Equal aliquots of lysates from Cos7 cells expressing Dvl1 alone (lane 2), myc-Daple alone (lane 3), or coexpressing both (lanes 1, 4) were used as source of Daple and Dvl in GST pulldown assays with recombinant, immobilized GST or GST-FZD7-CT. Bound proteins were analyzed for Dvl1 and Daple by immunoblotting (IB). Binding of each protein was higher when expressed alone (lanes 2, 3) than when co-expressed (lane 4). ( B ) Dvl loses colocalization with FZD7R at the PM after Wnt5a stimulation. HEK293 cells expressing FZD7-CFP were grown on cover slips coated with Poly-D-Lysine, starved overnight, and treated with 0.1 mg/ml Wnt5a as in 4B. Cells were fixed and stained for endogenous Dvl (red) and analyzed by confocal microscopy. ( C , D ) Generation and characterization of DLD1 7TGP cell lines stably expressing Daple. ( C ) DLD1 7TGP cell lines stably expressing Daple-WT or FA were starved and stimulated analyzed for Daple expression and phosphorylation of Akt by immunoblotting (IB). ( D ) Images display representative fields from monolayers of DLD1 cells grown in 0.2% FBS by fluorescence microscopy. The intensity of eGFP signals denotes Wnt transcriptional activity. Inset shows immunoblots (IB) of equal aliquots of whole-cell lysates of DLD1-7TGP cells expressing control vector, Daple-WT, or Daple-FA. Compared to DLD1 cells expressing Daple-WT, those expressing Daple-FA also express higher levels of GFP protein, indicative of higher Wnt transcriptional activity. DOI: http://dx.doi.org/10.7554/eLife.07091.012

    Article Snippet: Rabbit anti-pan-Gβ (M-14), anti-Gαi3, anti-DVL, and anti-β-catenin were obtained from Santa Cruz Biotechnology (Dallas, TX); anti-Akt and phospho-Akt (S473) were obtained from Cell Signaling; anti-Rac1 was obtained from BD Transduction Laboratories (San Jose, CA).

    Techniques: Binding Assay, Expressing, Recombinant, Staining, Confocal Microscopy, Stable Transfection, Fluorescence, Microscopy, Activity Assay, Western Blot, Plasmid Preparation

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: GSK-3β/Rac-1 pathway involved in sFRP-1-induced EC spreading. a: HUVECs were either infected with adenovirus expressing β-galactosidase, GSK-3β, GSK-3β-S9A, or GSK-3β-KM or treated with SB216763. After the treatment,

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Infection, Expressing

    sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: sFRP-1 induction of EC spreading is independent of β-catenin and requires GSK-3β activation. Endothelial cell lines expressing β-catenin (+/+) or deleted for β-catenin (−/−) as demonstrated

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Journal:

    Article Title: Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway

    doi: 10.2353/ajpath.2008.070130

    Figure Lengend Snippet: Role of sFRP-1 in neovessel formation and on the level of phosphorylated GSK-3β and β-catenin after hindlimb ischemia. a: Quantitative evaluation of capillary density using CD31 immunostaining (number of vessels/mm 2 ) in tissues retrieved

    Article Snippet: Membranes were incubated with the following antibodies: for the integrin activation, anti-phospho α-PAK and anti-α-PAK (Santa Cruz Biotechnology); for the Wnt/Frizzled pathway: anti-phospho-Ser9-GSK-3β (Cell Signaling), anti-total-GSK-3β (Cell Signaling), anti-β-catenin (Sigma), anti-phospho-ser9-β-catenin antibodies (Cell Signaling), anti-α tubulin (Sigma), anti-phospho-AKT (Cell Signaling) and anti-AKT (Cell Signaling); for Rac expression: anti-Rac1 (Pierce); for recombinant protein experiments, anti-His (Clontech), anti-Myc (Santa Cruz Biotechnology); for angiogenic factor expression: polyclonal antibodies against vascular endothelial growth factor, angiopoietin-1/2 (Santa Cruz Biotechnology).

    Techniques: Immunostaining

    Cleavage of endogenous Rac GTPases by recombinant caspase 3 in vitro. Whole-cell extracts from healthy JLP-119 cells containing endogenous Rac GTPases were incubated for different times at 37°C with purified recombinant caspase 3 or 8 at final concentrations of 5 ng/μl in a caspase reaction buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% (wt/vol) sucrose, and 10 mM dithiothreitol. Proteins were examined by Western blot immunoassay with the indicated antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Caspase 3-Mediated Inactivation of Rac GTPases Promotes Drug-Induced Apoptosis in Human Lymphoma Cells

    doi: 10.1128/MCB.23.16.5716-5725.2003

    Figure Lengend Snippet: Cleavage of endogenous Rac GTPases by recombinant caspase 3 in vitro. Whole-cell extracts from healthy JLP-119 cells containing endogenous Rac GTPases were incubated for different times at 37°C with purified recombinant caspase 3 or 8 at final concentrations of 5 ng/μl in a caspase reaction buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% (wt/vol) sucrose, and 10 mM dithiothreitol. Proteins were examined by Western blot immunoassay with the indicated antibodies.

    Article Snippet: For in vitro cleavage assays, recombinant His-Rac1 protein (100 ng/μl) was incubated with purified caspase 3 or 8 at a concentration of 5 ng/μl at 37°C in caspase reaction buffer (50 mM HEPES [pH 7.5], 100 mM NaCl, 1 mM EDTA, 10% [wt/vol] sucrose, 0.1% CHAPS, 10 mM dithiothreitol) (BD Biosciences).

    Techniques: Recombinant, In Vitro, Incubation, Purification, Western Blot