gtp rac1 pull down assay  (Millipore)


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    Structured Review

    Millipore gtp rac1 pull down assay
    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of <t>GTP</t> (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of <t>Rac1</t> GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value
    Gtp Rac1 Pull Down Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8638

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

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

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    Article Title: CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma
    Article Snippet: .. GTP-RAS and GTP-RAC1 pull-down assay The pull-down of GTP-bound RAS and RAC1 was performed by the use of active RAS and RAC1 pull-down and detection kits (Millipore) respectively according to the manufacturer's instructions. ..

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    Millipore gtp rac1 pull down assay
    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of <t>GTP</t> (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of <t>Rac1</t> GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value
    Gtp Rac1 Pull Down Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gtp rac1 pull down assay/product/Millipore
    Average 90 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    gtp rac1 pull down assay - by Bioz Stars, 2020-09
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    Millipore gtp bound rac1 cdc42
    Overexpression of Dsg3 in A431 cells significantly enhances the activity of <t>Rac1</t> and <t>Cdc42.</t> (A) Glutathione–sepharose beads complexed with GST-Pak-PBD or GST-Rhotekin-PBD fusion proteins were used to pull down the active <t>GTP-bound</t> Rac1 and Cdc42 or RhoA from lysates of A431-V and -D3 cells. Bead-bound complexes were loaded onto a 4–12% gradient gel and the amount of activated GTPases was determined by Western blotting with antibodies against Rac1, Cdc42 and RhoA. The negative control of GST pull down is displayed on the left. Significantly enhanced activity of Rac1 and Cdc42 but to a small extent, RhoA was detected in Dsg3 overexpressing cells. No evident changes were seen for total GTPases. Note that increased Dsg3 proteins were co-purified with the GTP-bound GTPases in D3 compared to V cells in both pull down assays. (B) Phase contrast images of A431-V and -D3 cells showing enhanced membrane projections and ruffling in D3 compared to V cells. Bar, 20 mm. (C) Graph showing ruffling velocity of A431-V and -C7 cells, treated in the absence or presence of the Rac1 inhibitor, NSC23766 at the concentration of 30 μM. Velocity measurements were obtained from kymograph analysis of the cell membrane. Overexpression of Dsg3 was shown to enhanced membrane ruffling and this could be blocked completely by Rac1 inhibitor. Data are collected from 24 regions of interest from 8 cells (pooled from 3 independent experiments, *** p
    Gtp Bound Rac1 Cdc42, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of GTP (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of Rac1 GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.8638

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

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

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

    The effects of PTP1B inhibitor on EC motility were dependent on Rac1. ( A ) The effects of PTPI22 on cell spreading in the absence or presence of the Rac1 inhibitor NSC23766 (100 μM). ( B ) GTPase pull-down assay showing that PTPI22 treatment increased the amount of GTP-bound Rac1 in sub-confluent TIME cells. ( C , D ) Effects of the Rho inhibitor Rhosin (1 μM) on PTPI22-stimulated cell spreading response. The presence of stress fibres in untreated cells was indicated by white arrowheads; Rhosin decreased the abundance of intracellular stress fibres. ( E , F ) Effects of NSC23766 and Rhosin on PTPI22-stimulated EC migration assessed by the Boyden chamber assay. Data were mean ± S.E.M.; * P

    Journal: Scientific Reports

    Article Title: PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway

    doi: 10.1038/srep24111

    Figure Lengend Snippet: The effects of PTP1B inhibitor on EC motility were dependent on Rac1. ( A ) The effects of PTPI22 on cell spreading in the absence or presence of the Rac1 inhibitor NSC23766 (100 μM). ( B ) GTPase pull-down assay showing that PTPI22 treatment increased the amount of GTP-bound Rac1 in sub-confluent TIME cells. ( C , D ) Effects of the Rho inhibitor Rhosin (1 μM) on PTPI22-stimulated cell spreading response. The presence of stress fibres in untreated cells was indicated by white arrowheads; Rhosin decreased the abundance of intracellular stress fibres. ( E , F ) Effects of NSC23766 and Rhosin on PTPI22-stimulated EC migration assessed by the Boyden chamber assay. Data were mean ± S.E.M.; * P

    Article Snippet: Before experimentation, cells were washed with serum free medium, and then incubated with vehicle or 10 μM PTPI22 for different for 20 or 40 min. Pull-down assay for GTP-bound Rac1 was performed using a Rac1 Activation Assay Kit from EMD Millipore (Billerica, MA, USA).

    Techniques: Pull Down Assay, Migration, Boyden Chamber Assay

    The lipid binding deficient mutant of neurofibromin 2 displays impaired inhibition of Rac1 activation and YAP activity. a 293 T or b SC4 cells were transfected with expression vectors for wild type or lipid binding deficient neurofibromin 2 or empty vector control (pCDNA) and levels of active Rac1 (Rac1-GTP) were assessed after 48 h. Levels of total Rac1, neurofibromin 2, and tubulin were assessed as controls. The blots shown are representative of three biological replicates. c HEK293T cells were transfected with expression vectors for wild type or lipid binding deficient neurofibromin 2 or empty vector control (pCDNA) along with YAP-driven luciferase and Renilla luciferase reporters. Activity of the luciferase reporter was assessed 24 h post transfection. Means of each data point were calculated from three independent biological replicates conducted in triplicate. Error bars represent ± S.D

    Journal: Nature Communications

    Article Title: Lipid binding promotes the open conformation and tumor-suppressive activity of neurofibromin 2

    doi: 10.1038/s41467-018-03648-4

    Figure Lengend Snippet: The lipid binding deficient mutant of neurofibromin 2 displays impaired inhibition of Rac1 activation and YAP activity. a 293 T or b SC4 cells were transfected with expression vectors for wild type or lipid binding deficient neurofibromin 2 or empty vector control (pCDNA) and levels of active Rac1 (Rac1-GTP) were assessed after 48 h. Levels of total Rac1, neurofibromin 2, and tubulin were assessed as controls. The blots shown are representative of three biological replicates. c HEK293T cells were transfected with expression vectors for wild type or lipid binding deficient neurofibromin 2 or empty vector control (pCDNA) along with YAP-driven luciferase and Renilla luciferase reporters. Activity of the luciferase reporter was assessed 24 h post transfection. Means of each data point were calculated from three independent biological replicates conducted in triplicate. Error bars represent ± S.D

    Article Snippet: Immunoprecipitation and Rac1-GTP pull-down Cells were transfected with 8 μg of plasmid DNA (empty vector control or different neurofibromin 2 alleles) and Rac1-GTP levels were determined using a pull-down approach employing a fusion protein comprised of the p21-binding domain of PAK1 fused to GST, according to the manufacturer’s instructions (Millipore, #17-441).

    Techniques: Binding Assay, Mutagenesis, Inhibition, Activation Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase

    Overexpression of Dsg3 in A431 cells significantly enhances the activity of Rac1 and Cdc42. (A) Glutathione–sepharose beads complexed with GST-Pak-PBD or GST-Rhotekin-PBD fusion proteins were used to pull down the active GTP-bound Rac1 and Cdc42 or RhoA from lysates of A431-V and -D3 cells. Bead-bound complexes were loaded onto a 4–12% gradient gel and the amount of activated GTPases was determined by Western blotting with antibodies against Rac1, Cdc42 and RhoA. The negative control of GST pull down is displayed on the left. Significantly enhanced activity of Rac1 and Cdc42 but to a small extent, RhoA was detected in Dsg3 overexpressing cells. No evident changes were seen for total GTPases. Note that increased Dsg3 proteins were co-purified with the GTP-bound GTPases in D3 compared to V cells in both pull down assays. (B) Phase contrast images of A431-V and -D3 cells showing enhanced membrane projections and ruffling in D3 compared to V cells. Bar, 20 mm. (C) Graph showing ruffling velocity of A431-V and -C7 cells, treated in the absence or presence of the Rac1 inhibitor, NSC23766 at the concentration of 30 μM. Velocity measurements were obtained from kymograph analysis of the cell membrane. Overexpression of Dsg3 was shown to enhanced membrane ruffling and this could be blocked completely by Rac1 inhibitor. Data are collected from 24 regions of interest from 8 cells (pooled from 3 independent experiments, *** p

    Journal: Experimental Cell Research

    Article Title: Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

    doi: 10.1016/j.yexcr.2012.07.002

    Figure Lengend Snippet: Overexpression of Dsg3 in A431 cells significantly enhances the activity of Rac1 and Cdc42. (A) Glutathione–sepharose beads complexed with GST-Pak-PBD or GST-Rhotekin-PBD fusion proteins were used to pull down the active GTP-bound Rac1 and Cdc42 or RhoA from lysates of A431-V and -D3 cells. Bead-bound complexes were loaded onto a 4–12% gradient gel and the amount of activated GTPases was determined by Western blotting with antibodies against Rac1, Cdc42 and RhoA. The negative control of GST pull down is displayed on the left. Significantly enhanced activity of Rac1 and Cdc42 but to a small extent, RhoA was detected in Dsg3 overexpressing cells. No evident changes were seen for total GTPases. Note that increased Dsg3 proteins were co-purified with the GTP-bound GTPases in D3 compared to V cells in both pull down assays. (B) Phase contrast images of A431-V and -D3 cells showing enhanced membrane projections and ruffling in D3 compared to V cells. Bar, 20 mm. (C) Graph showing ruffling velocity of A431-V and -C7 cells, treated in the absence or presence of the Rac1 inhibitor, NSC23766 at the concentration of 30 μM. Velocity measurements were obtained from kymograph analysis of the cell membrane. Overexpression of Dsg3 was shown to enhanced membrane ruffling and this could be blocked completely by Rac1 inhibitor. Data are collected from 24 regions of interest from 8 cells (pooled from 3 independent experiments, *** p

    Article Snippet: For pull down assay using GTPases as baits, glutathione–agarose beads complexed with the GST fusion proteins, corresponding to the p21-binding domain (PBD) of human PAK-1 or Rhotekin, were used to pull down the active GTP-bound Rac1/Cdc42 and RhoA from A431-V and -D3 cells following the manufacturing protocol (Rac1/Cdc42 Activation Assay Kit, Millipore).

    Techniques: Over Expression, Activity Assay, Western Blot, Negative Control, Purification, Concentration Assay