e 1000 2  (Echelon Biosciences)


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    Echelon Biosciences e 1000 2
    E 1000 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    e 1000 2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences e 1000 2
    E 1000 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal anti ship2  (Echelon Biosciences)


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    Echelon Biosciences polyclonal anti ship2
    Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, <t>anti-SHIP2,</t> and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.
    Polyclonal Anti Ship2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti ship2/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti ship2 - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration"

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E11-11-0958

    Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, anti-SHIP2, and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.
    Figure Legend Snippet: Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, anti-SHIP2, and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Techniques Used: Binding Assay, Isolation, Affinity Column, Chromatography, Incubation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing

    Interaction of SHIP2 with active RhoA. (A) Domain structure and deletion constructs of SHIP2. (B) Mapping of the SHIP2 region required for binding to active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with COS7 cell lysate expressing GFP-SHIP2-N, GFP-SHIP2-cat, or GFP-SHIP2-C. The bound proteins were analyzed by immunoblotting with the anti-GFP antibody. (C) Direct binding of SHIP2 with active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with various MBP-SHIP2 fragments. The bound proteins were analyzed by silver staining. Black dots indicate respective intact bands, and arrowheads indicate the bound proteins. (D) Characterization of binding-deficient mutants of SHIP2 in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2-WT or myc-SHIP2-D193/E195A and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.
    Figure Legend Snippet: Interaction of SHIP2 with active RhoA. (A) Domain structure and deletion constructs of SHIP2. (B) Mapping of the SHIP2 region required for binding to active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with COS7 cell lysate expressing GFP-SHIP2-N, GFP-SHIP2-cat, or GFP-SHIP2-C. The bound proteins were analyzed by immunoblotting with the anti-GFP antibody. (C) Direct binding of SHIP2 with active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with various MBP-SHIP2 fragments. The bound proteins were analyzed by silver staining. Black dots indicate respective intact bands, and arrowheads indicate the bound proteins. (D) Characterization of binding-deficient mutants of SHIP2 in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2-WT or myc-SHIP2-D193/E195A and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Techniques Used: Construct, Binding Assay, Incubation, Expressing, Western Blot, Silver Staining

    Association of SHIP2 with RhoA in intact cells. (A) Localization of SHIP2 and RhoA in U251 cells. U251 cells were double labeled with anti-SHIP2 (green) and anti-RhoA (red) antibodies. Right, the merged image. Bar, 10 μm. (B) Association of SHIP2 with RhoA by proximity ligation assays in U251 cells. U251 cells were treated with control siRNA or siRNA against RhoA. PLA was performed using antibodies against SHIP2, RhoA, or both. Images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked with a dotted line. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar, 10 μm. (C) Quantification of PLAs. Quantification of PLAs conducted in B. Asterisks indicate a difference from the value of SHIP2/RhoA PLA cells at p < 0.01. Error bars indicate ± SD. (D) Association of SHIP2 with RhoA by PLA in spreading U251 cells. U251 cells were suspended and seeded on FN-coated glasses and fixed at 30 min and 1, 2, 4, and, 8 h after plating. PLA was performed using antibodies against both SHIP2 and RhoA. Cells were stained with anti-PI(3,4,5)P 3 , anti-RhoA, and anti-SHIP2 antibodies. PLA images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked by a dotted line. Nuclei were stained with DAPI (blue). Bar, 10 μm. All results are representative of at least three independent experiments. At least 50 cells were counted per experiment.
    Figure Legend Snippet: Association of SHIP2 with RhoA in intact cells. (A) Localization of SHIP2 and RhoA in U251 cells. U251 cells were double labeled with anti-SHIP2 (green) and anti-RhoA (red) antibodies. Right, the merged image. Bar, 10 μm. (B) Association of SHIP2 with RhoA by proximity ligation assays in U251 cells. U251 cells were treated with control siRNA or siRNA against RhoA. PLA was performed using antibodies against SHIP2, RhoA, or both. Images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked with a dotted line. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar, 10 μm. (C) Quantification of PLAs. Quantification of PLAs conducted in B. Asterisks indicate a difference from the value of SHIP2/RhoA PLA cells at p < 0.01. Error bars indicate ± SD. (D) Association of SHIP2 with RhoA by PLA in spreading U251 cells. U251 cells were suspended and seeded on FN-coated glasses and fixed at 30 min and 1, 2, 4, and, 8 h after plating. PLA was performed using antibodies against both SHIP2 and RhoA. Cells were stained with anti-PI(3,4,5)P 3 , anti-RhoA, and anti-SHIP2 antibodies. PLA images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked by a dotted line. Nuclei were stained with DAPI (blue). Bar, 10 μm. All results are representative of at least three independent experiments. At least 50 cells were counted per experiment.

    Techniques Used: Labeling, Ligation, Staining

    Requirement of SHIP2 for proper polarization in U251 cells. (A) Effect of the depletion of SHIP2 and RhoA in U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, fixed, and stained with tetramethylrhodamine isothiocyanate (TRITC)–phalloidin. Bar, 10 μm. (B) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with TRITC–phalloidin and the anti-GFP antibody. White arrows indicate transfected cells. Bar, 20 μm. (C) Quantification of the effects of SHIP2 and RhoA mutants on cell polarity. The percentage of the cells that had a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.
    Figure Legend Snippet: Requirement of SHIP2 for proper polarization in U251 cells. (A) Effect of the depletion of SHIP2 and RhoA in U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, fixed, and stained with tetramethylrhodamine isothiocyanate (TRITC)–phalloidin. Bar, 10 μm. (B) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with TRITC–phalloidin and the anti-GFP antibody. White arrows indicate transfected cells. Bar, 20 μm. (C) Quantification of the effects of SHIP2 and RhoA mutants on cell polarity. The percentage of the cells that had a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Techniques Used: Transfection, Staining

    Requirement of SHIP2 for the proper accumulation of PI(3,4,5)P 3 . (A) PI(3,4,5)P 3 localization in SHIP2- or RhoA-depleted U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, cultured for 1 h in the presence or absence of 50 μM LY294002, fixed, and stained with Alexa 488–phalloidin and the anti-PI(3,4,5)P 3 antibody. Bar, 10 μm. (B) Quantification of the effects of SHIP2 or RhoA knockdown on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. (C) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with anti-PI(3,4,5)P 3 and anti-GFP antibodies. White arrows indicate transfected cells. Bar, 20 μm (D) Quantification of the effects of SHIP2 knockdown and rescue mutants on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.
    Figure Legend Snippet: Requirement of SHIP2 for the proper accumulation of PI(3,4,5)P 3 . (A) PI(3,4,5)P 3 localization in SHIP2- or RhoA-depleted U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, cultured for 1 h in the presence or absence of 50 μM LY294002, fixed, and stained with Alexa 488–phalloidin and the anti-PI(3,4,5)P 3 antibody. Bar, 10 μm. (B) Quantification of the effects of SHIP2 or RhoA knockdown on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. (C) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with anti-PI(3,4,5)P 3 and anti-GFP antibodies. White arrows indicate transfected cells. Bar, 20 μm (D) Quantification of the effects of SHIP2 knockdown and rescue mutants on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Techniques Used: Transfection, Cell Culture, Staining

    Requirement of the RhoA-binding activity of SHIP2 for cell migration. U251 cells transfected with siRNA along with the indicated plasmids were subjected to the Boyden chamber assay. The cells were allowed to migrate for 2 h. Cells were fixed and stained with the anti-GFP antibody. All results are representative of at least three independent experiments. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. At least 300 EGFP-positive cells were counted per experiment.
    Figure Legend Snippet: Requirement of the RhoA-binding activity of SHIP2 for cell migration. U251 cells transfected with siRNA along with the indicated plasmids were subjected to the Boyden chamber assay. The cells were allowed to migrate for 2 h. Cells were fixed and stained with the anti-GFP antibody. All results are representative of at least three independent experiments. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. At least 300 EGFP-positive cells were counted per experiment.

    Techniques Used: Binding Assay, Activity Assay, Migration, Transfection, Boyden Chamber Assay, Staining

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    Echelon Biosciences e 1000 2
    E 1000 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    Echelon Biosciences polyclonal anti ship2
    Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, <t>anti-SHIP2,</t> and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.
    Polyclonal Anti Ship2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti ship2/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti ship2 - by Bioz Stars, 2023-02
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    Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, anti-SHIP2, and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Identification of RhoA-binding proteins. (A) Isolation of RhoA-binding proteins by affinity column chromatography. GST, GDP⋅GST-RhoA, GTPγS⋅GST-RhoA, or GST-RhoA mutants were immobilized on beads and incubated with the rat heart lysate. The bound proteins were eluted with buffer containing 1 M NaCl and subjected to SDS–PAGE, followed by silver staining. (B) Validation of LC-MS/MS results by immunoblotting. The eluates from affinity column chromatography were subjected to immunoblotting using anti–Rho-kinase, anti-RhoGDI, anti-SHIP2, and anti-ACAT1 antibodies. (C) Interaction of SHIP2 with active RhoA in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The dashed line indicates separate membranes. (D) Specific interaction of SHIP2 with RhoA but not Rac1 or Cdc42. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2 and GST-RhoA, GST-Rac1, or GST-Cdc42 mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Binding Assay, Isolation, Affinity Column, Chromatography, Incubation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing

    Interaction of SHIP2 with active RhoA. (A) Domain structure and deletion constructs of SHIP2. (B) Mapping of the SHIP2 region required for binding to active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with COS7 cell lysate expressing GFP-SHIP2-N, GFP-SHIP2-cat, or GFP-SHIP2-C. The bound proteins were analyzed by immunoblotting with the anti-GFP antibody. (C) Direct binding of SHIP2 with active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with various MBP-SHIP2 fragments. The bound proteins were analyzed by silver staining. Black dots indicate respective intact bands, and arrowheads indicate the bound proteins. (D) Characterization of binding-deficient mutants of SHIP2 in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2-WT or myc-SHIP2-D193/E195A and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Interaction of SHIP2 with active RhoA. (A) Domain structure and deletion constructs of SHIP2. (B) Mapping of the SHIP2 region required for binding to active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with COS7 cell lysate expressing GFP-SHIP2-N, GFP-SHIP2-cat, or GFP-SHIP2-C. The bound proteins were analyzed by immunoblotting with the anti-GFP antibody. (C) Direct binding of SHIP2 with active RhoA. GST, GDP⋅GST-RhoA, or GTPγS⋅GST-RhoA, immobilized on beads, was incubated with various MBP-SHIP2 fragments. The bound proteins were analyzed by silver staining. Black dots indicate respective intact bands, and arrowheads indicate the bound proteins. (D) Characterization of binding-deficient mutants of SHIP2 in COS7 cells. Glutathione–Sepharose beads were incubated with COS7 cell lysate expressing both myc-SHIP2-WT or myc-SHIP2-D193/E195A and GST-RhoA mutants. The bound proteins were analyzed by immunoblotting with anti-myc and anti-GST antibodies. All results are representative of at least three independent experiments.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Construct, Binding Assay, Incubation, Expressing, Western Blot, Silver Staining

    Association of SHIP2 with RhoA in intact cells. (A) Localization of SHIP2 and RhoA in U251 cells. U251 cells were double labeled with anti-SHIP2 (green) and anti-RhoA (red) antibodies. Right, the merged image. Bar, 10 μm. (B) Association of SHIP2 with RhoA by proximity ligation assays in U251 cells. U251 cells were treated with control siRNA or siRNA against RhoA. PLA was performed using antibodies against SHIP2, RhoA, or both. Images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked with a dotted line. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar, 10 μm. (C) Quantification of PLAs. Quantification of PLAs conducted in B. Asterisks indicate a difference from the value of SHIP2/RhoA PLA cells at p < 0.01. Error bars indicate ± SD. (D) Association of SHIP2 with RhoA by PLA in spreading U251 cells. U251 cells were suspended and seeded on FN-coated glasses and fixed at 30 min and 1, 2, 4, and, 8 h after plating. PLA was performed using antibodies against both SHIP2 and RhoA. Cells were stained with anti-PI(3,4,5)P 3 , anti-RhoA, and anti-SHIP2 antibodies. PLA images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked by a dotted line. Nuclei were stained with DAPI (blue). Bar, 10 μm. All results are representative of at least three independent experiments. At least 50 cells were counted per experiment.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Association of SHIP2 with RhoA in intact cells. (A) Localization of SHIP2 and RhoA in U251 cells. U251 cells were double labeled with anti-SHIP2 (green) and anti-RhoA (red) antibodies. Right, the merged image. Bar, 10 μm. (B) Association of SHIP2 with RhoA by proximity ligation assays in U251 cells. U251 cells were treated with control siRNA or siRNA against RhoA. PLA was performed using antibodies against SHIP2, RhoA, or both. Images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked with a dotted line. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar, 10 μm. (C) Quantification of PLAs. Quantification of PLAs conducted in B. Asterisks indicate a difference from the value of SHIP2/RhoA PLA cells at p < 0.01. Error bars indicate ± SD. (D) Association of SHIP2 with RhoA by PLA in spreading U251 cells. U251 cells were suspended and seeded on FN-coated glasses and fixed at 30 min and 1, 2, 4, and, 8 h after plating. PLA was performed using antibodies against both SHIP2 and RhoA. Cells were stained with anti-PI(3,4,5)P 3 , anti-RhoA, and anti-SHIP2 antibodies. PLA images represent focused views of several confocal sections that covered the entire region of cells. Cell edges are marked by a dotted line. Nuclei were stained with DAPI (blue). Bar, 10 μm. All results are representative of at least three independent experiments. At least 50 cells were counted per experiment.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Labeling, Ligation, Staining

    Requirement of SHIP2 for proper polarization in U251 cells. (A) Effect of the depletion of SHIP2 and RhoA in U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, fixed, and stained with tetramethylrhodamine isothiocyanate (TRITC)–phalloidin. Bar, 10 μm. (B) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with TRITC–phalloidin and the anti-GFP antibody. White arrows indicate transfected cells. Bar, 20 μm. (C) Quantification of the effects of SHIP2 and RhoA mutants on cell polarity. The percentage of the cells that had a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Requirement of SHIP2 for proper polarization in U251 cells. (A) Effect of the depletion of SHIP2 and RhoA in U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, fixed, and stained with tetramethylrhodamine isothiocyanate (TRITC)–phalloidin. Bar, 10 μm. (B) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with TRITC–phalloidin and the anti-GFP antibody. White arrows indicate transfected cells. Bar, 20 μm. (C) Quantification of the effects of SHIP2 and RhoA mutants on cell polarity. The percentage of the cells that had a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Transfection, Staining

    Requirement of SHIP2 for the proper accumulation of PI(3,4,5)P 3 . (A) PI(3,4,5)P 3 localization in SHIP2- or RhoA-depleted U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, cultured for 1 h in the presence or absence of 50 μM LY294002, fixed, and stained with Alexa 488–phalloidin and the anti-PI(3,4,5)P 3 antibody. Bar, 10 μm. (B) Quantification of the effects of SHIP2 or RhoA knockdown on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. (C) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with anti-PI(3,4,5)P 3 and anti-GFP antibodies. White arrows indicate transfected cells. Bar, 20 μm (D) Quantification of the effects of SHIP2 knockdown and rescue mutants on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Requirement of SHIP2 for the proper accumulation of PI(3,4,5)P 3 . (A) PI(3,4,5)P 3 localization in SHIP2- or RhoA-depleted U251 cells. U251 cells transfected with the indicated siRNAs for 72 h were reseeded on FN-coated glasses for 8 h, cultured for 1 h in the presence or absence of 50 μM LY294002, fixed, and stained with Alexa 488–phalloidin and the anti-PI(3,4,5)P 3 antibody. Bar, 10 μm. (B) Quantification of the effects of SHIP2 or RhoA knockdown on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. (C) Rescue experiments of SHIP2 knockdown in U251 cells. U251 cells transfected with both the indicated siRNAs and plasmids were reseeded on FN-coated glasses for 8 h, fixed, and stained with anti-PI(3,4,5)P 3 and anti-GFP antibodies. White arrows indicate transfected cells. Bar, 20 μm (D) Quantification of the effects of SHIP2 knockdown and rescue mutants on the accumulation of PI(3,4,5)P 3 at the leading edge. The percentage of the cells in which the accumulation of PI(3,4,5)P 3 was observed at a single leading edge is shown. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. All results are representative of at least three independent experiments. At least 100 cells were counted per experiment.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Transfection, Cell Culture, Staining

    Requirement of the RhoA-binding activity of SHIP2 for cell migration. U251 cells transfected with siRNA along with the indicated plasmids were subjected to the Boyden chamber assay. The cells were allowed to migrate for 2 h. Cells were fixed and stained with the anti-GFP antibody. All results are representative of at least three independent experiments. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. At least 300 EGFP-positive cells were counted per experiment.

    Journal: Molecular Biology of the Cell

    Article Title: The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

    doi: 10.1091/mbc.E11-11-0958

    Figure Lengend Snippet: Requirement of the RhoA-binding activity of SHIP2 for cell migration. U251 cells transfected with siRNA along with the indicated plasmids were subjected to the Boyden chamber assay. The cells were allowed to migrate for 2 h. Cells were fixed and stained with the anti-GFP antibody. All results are representative of at least three independent experiments. Asterisks indicate a difference from the value of control cells at p < 0.01. Error bars indicate ± SD. At least 300 EGFP-positive cells were counted per experiment.

    Article Snippet: The antibodies used were as follows: monoclonal anti-GFP (Roche Diagnostics, Mannheim, Germany); polyclonal anti-GFP (MBL, Nagoya, Japan); monoclonal anti-SHIP2 and anti-HA (Cell Signaling Technology, Beverly, MA); polyclonal anti-INPPL1 (Abcam, Cambridge, United Kingdom); monoclonal anti-PI(3,4,5)P 3 (Echelon Bioscience, Salt Lake City, UT); monoclonal anti-RhoA and anti-SHIP1, polyclonal anti-SHIP2 and anti-RhoGDI (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal anti-ACAT1 (GeneTex, Irvine, CA); monoclonal anti–α-tubulin (Sigma-Aldrich, St. Louis, MO); monoclonal anti-myc (Wako Pure Chemical Industries, Osaka, Japan); and monoclonal anti–N-cadherin (BD Biosciences, San Jose, CA).

    Techniques: Binding Assay, Activity Assay, Migration, Transfection, Boyden Chamber Assay, Staining