phospho-mek1 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Thermo Fisher phospho mek1 ser298 polyclonal antibody
    Phospho Mek1 Ser298 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1 ser298 polyclonal antibody/product/Thermo Fisher
    Average 92 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 ser298 polyclonal antibody - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    98
    Millipore phospho mek1
    CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins <t>(MEK1).</t> Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p
    Phospho Mek1, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1/product/Millipore
    Average 98 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 - by Bioz Stars, 2020-08
    98/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc phospho mek1
    A : NES-B3T cells have higher levels of <t>MEK1</t> phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in
    Phospho Mek1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1/product/Cell Signaling Technology Inc
    Average 95 stars, based on 64 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc phospho mek1 ser298
    PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1 −/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1 −/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1 −/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and <t>phospho-Ser298-MEK1/2</t> levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×10 5 Wt or PAK1 −/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.
    Phospho Mek1 Ser298, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1 ser298/product/Cell Signaling Technology Inc
    Average 93 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 ser298 - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho mek1 2ser217 221
    PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1 −/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1 −/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1 −/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and <t>phospho-Ser298-MEK1/2</t> levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×10 5 Wt or PAK1 −/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.
    Phospho Mek1 2ser217 221, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1 2ser217 221/product/Cell Signaling Technology Inc
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 2ser217 221 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc pathscan phospho mek1
    PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1 −/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1 −/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1 −/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and <t>phospho-Ser298-MEK1/2</t> levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×10 5 Wt or PAK1 −/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.
    Pathscan Phospho Mek1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pathscan phospho mek1/product/Cell Signaling Technology Inc
    Average 90 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    pathscan phospho mek1 - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    88
    Thermo Fisher phospho mek1 2
    DA-Raf-dependent inactivation of <t>MEK1/2</t> suppresses TIMP4 expression and induces a subsequent proteolytic cascade MMP14–MMP2. ( A ) Total lungs and AEC2s were collected from X/Y (pink boxes) and X – /Y (blue boxes) mice at P5, and the expression levels of Timp4 were analyzed by using real-time PCR. ( B ) Lung lysates isolated from X/Y and X – /Y mice were subjected to the gelatin zymography analysis. ( C ) Activity levels of MMP14 in the lungs of X/Y (pink bar) and X – . The values represent means ± SD. ( D ) The expression levels of Mmp2 , Mmp9 , and Mmp14 in lungs at P5 from X/Y and X – /Y mice were analyzed by real-time PCR. Values represent the amounts of mRNA relative to those in WT littermates, which are arbitrarily defined as 1. ( E ) MMP2 activity ( Top ) was analyzed by zymography using lysates from developing lungs. The amounts of TIMP4 ( Middle ) and β-Tubulin ( Bottom ) were examined by Western blot analysis at indicated postnatal ages. ( F and G ) The expression levels of Timp4 in lungs at P6 from X/Y and X – /Y mice treated with DMSO or MEKi were examined by using real-time PCR ( F ) or Western blot analysis ( G ). ( H ) MMP2 activity levels were analyzed gelatin zymography by use of lung lysates isolated from X/Y and X – /Y mice treated with DMSO or the MEKi. In A , C , D , and F the analyses were performed with three to five independent mice per genotype. ** P
    Phospho Mek1 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho mek1 2/product/Thermo Fisher
    Average 88 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 2 - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc mek1 phospho mek1 elisa kit
    DA-Raf-dependent inactivation of <t>MEK1/2</t> suppresses TIMP4 expression and induces a subsequent proteolytic cascade MMP14–MMP2. ( A ) Total lungs and AEC2s were collected from X/Y (pink boxes) and X – /Y (blue boxes) mice at P5, and the expression levels of Timp4 were analyzed by using real-time PCR. ( B ) Lung lysates isolated from X/Y and X – /Y mice were subjected to the gelatin zymography analysis. ( C ) Activity levels of MMP14 in the lungs of X/Y (pink bar) and X – . The values represent means ± SD. ( D ) The expression levels of Mmp2 , Mmp9 , and Mmp14 in lungs at P5 from X/Y and X – /Y mice were analyzed by real-time PCR. Values represent the amounts of mRNA relative to those in WT littermates, which are arbitrarily defined as 1. ( E ) MMP2 activity ( Top ) was analyzed by zymography using lysates from developing lungs. The amounts of TIMP4 ( Middle ) and β-Tubulin ( Bottom ) were examined by Western blot analysis at indicated postnatal ages. ( F and G ) The expression levels of Timp4 in lungs at P6 from X/Y and X – /Y mice treated with DMSO or MEKi were examined by using real-time PCR ( F ) or Western blot analysis ( G ). ( H ) MMP2 activity levels were analyzed gelatin zymography by use of lung lysates isolated from X/Y and X – /Y mice treated with DMSO or the MEKi. In A , C , D , and F the analyses were performed with three to five independent mice per genotype. ** P
    Mek1 Phospho Mek1 Elisa Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mek1 phospho mek1 elisa kit/product/Cell Signaling Technology Inc
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mek1 phospho mek1 elisa kit - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    85
    Thermo Fisher rabbit polyclonal anti phospho s222 mek1
    DA-Raf-dependent inactivation of <t>MEK1/2</t> suppresses TIMP4 expression and induces a subsequent proteolytic cascade MMP14–MMP2. ( A ) Total lungs and AEC2s were collected from X/Y (pink boxes) and X – /Y (blue boxes) mice at P5, and the expression levels of Timp4 were analyzed by using real-time PCR. ( B ) Lung lysates isolated from X/Y and X – /Y mice were subjected to the gelatin zymography analysis. ( C ) Activity levels of MMP14 in the lungs of X/Y (pink bar) and X – . The values represent means ± SD. ( D ) The expression levels of Mmp2 , Mmp9 , and Mmp14 in lungs at P5 from X/Y and X – /Y mice were analyzed by real-time PCR. Values represent the amounts of mRNA relative to those in WT littermates, which are arbitrarily defined as 1. ( E ) MMP2 activity ( Top ) was analyzed by zymography using lysates from developing lungs. The amounts of TIMP4 ( Middle ) and β-Tubulin ( Bottom ) were examined by Western blot analysis at indicated postnatal ages. ( F and G ) The expression levels of Timp4 in lungs at P6 from X/Y and X – /Y mice treated with DMSO or MEKi were examined by using real-time PCR ( F ) or Western blot analysis ( G ). ( H ) MMP2 activity levels were analyzed gelatin zymography by use of lung lysates isolated from X/Y and X – /Y mice treated with DMSO or the MEKi. In A , C , D , and F the analyses were performed with three to five independent mice per genotype. ** P
    Rabbit Polyclonal Anti Phospho S222 Mek1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti phospho s222 mek1/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti phospho s222 mek1 - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p

    Journal: Cancer discovery

    Article Title: Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers

    doi: 10.1158/2159-8290.CD-11-0046

    Figure Lengend Snippet: CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p

    Article Snippet: Beads were boiled in sample buffer for 5 min and supernatants were resolved for immunoblotting for phospho-MEK1 (#07-461, Millipore).

    Techniques: Transformation Assay, Over Expression, Activity Assay, Plasmid Preparation, Pull Down Assay, In Vitro, Expressing, Construct, Isolation, Immunoprecipitation, Western Blot, Kinase Assay, Mutagenesis, shRNA

    Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).

    Journal: The Journal of Cell Biology

    Article Title: PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

    doi: 10.1083/jcb.200212141

    Figure Lengend Snippet: Src inhibition alters PAK-mediated MEK phosphorylation. (A) REF52 cells were incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on 10 μg/ml FN for 5, 10, 20, or 40 min in the continued presence or absence of PP2. Whole cell lysates were blotted with p-S298MEK1, p-S218/S222MEK1, MEK1, p-MAPK, or ERK2 antisera. Densitometric analysis demonstrated that the intensity of p-S298MEK1 after a 5-min plating on FN in the presence of 50 μM PP2 was ∼28% the level of the DMSO-treated control when normalized to an MEK1 loading control. The observed decrease in p-MAPK levels in lane 9 was not reproducible. (B) REF52 cells were cotransfected with HA-tagged MEK1 constructs and PAK1 T423E or vector control, incubated in suspension with 50 μM PP2 or DMSO control (S) and plated on FN for 20 min. Anti-HA immunoprecipitates were formed and blotted with pS218/S222 antiserum (top), HA antiserum (second [from top] panel), and subsequently with anti-pS298MEK1 (third [from top] panel). Western blotting of lysates with anti-myc antiserum confirmed expression of activated PAK1 (bottom).

    Article Snippet: The following antibodies were used: phospho-MAPK (New England Biolabs, Inc.; ); MEK1/2 (New England Biolabs, Inc.); phospho-MEK1 S218/S222 (Sigma-Aldrich); ERK2 (clone B3B9; Upstate Biotechnology); MEK1, MEK2, and paxillin (Transduction Labs); HA (clone 12CA5); myc (clone 9E10; Transduction Labs); p-FAK Y397 (Biosource International); PAK (anti-N20 for immunoprecipitation; anti-C19 for Western analysis); c-Raf (anti–C-12); and Gal4 transactivation domain (Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition, Incubation, Construct, Plasmid Preparation, Western Blot, Expressing

    Phosphorylation of MEK1 on S298 regulates MEK1 activation. REF52 cells were transiently transfected with HA-MEK1, HA-MEK1 T292A, HA-MEK1 S298A, or HA-MEK1 T292A/S298A. Cells were suspended for 90 min (S) and plated on FN for 20 min (FN). Anti-HA immunoprecipitates were formed and blotted with anti-p-S218/S222MEK1 (top), anti-pS298MEK1 (middle), or anti-HA antiserum (bottom).

    Journal: The Journal of Cell Biology

    Article Title: PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

    doi: 10.1083/jcb.200212141

    Figure Lengend Snippet: Phosphorylation of MEK1 on S298 regulates MEK1 activation. REF52 cells were transiently transfected with HA-MEK1, HA-MEK1 T292A, HA-MEK1 S298A, or HA-MEK1 T292A/S298A. Cells were suspended for 90 min (S) and plated on FN for 20 min (FN). Anti-HA immunoprecipitates were formed and blotted with anti-p-S218/S222MEK1 (top), anti-pS298MEK1 (middle), or anti-HA antiserum (bottom).

    Article Snippet: The following antibodies were used: phospho-MAPK (New England Biolabs, Inc.; ); MEK1/2 (New England Biolabs, Inc.); phospho-MEK1 S218/S222 (Sigma-Aldrich); ERK2 (clone B3B9; Upstate Biotechnology); MEK1, MEK2, and paxillin (Transduction Labs); HA (clone 12CA5); myc (clone 9E10; Transduction Labs); p-FAK Y397 (Biosource International); PAK (anti-N20 for immunoprecipitation; anti-C19 for Western analysis); c-Raf (anti–C-12); and Gal4 transactivation domain (Santa Cruz Biotechnology, Inc.).

    Techniques: Activation Assay, Transfection

    Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

    doi: 10.1083/jcb.200212141

    Figure Lengend Snippet: Adhesion stimulates MAPK and MEK phosphorylation. REF52 cells were either continuously adherent (A) or suspended (S) and plated on FN for 5, 10, 20, or 40 min. Whole cell lysates were blotted with antiserum specific for (A) phosphorylated MAPK (p-MAPK; top) or ERK2 (bottom), or (B) MEK1 phosphorylated on S218/S222 (p-S218/222MEK1; top) or MEK1 (bottom). (C) REF52 cells were suspended for 1 h and plated on FN for 1 h before co-staining for p-MAPK (red) and paxillin (green). The arrows indicate focal complex-like structures containing p-MAPK; arrowheads indicate paxillin-containing focal adhesions. Bar, 10 μm.

    Article Snippet: The following antibodies were used: phospho-MAPK (New England Biolabs, Inc.; ); MEK1/2 (New England Biolabs, Inc.); phospho-MEK1 S218/S222 (Sigma-Aldrich); ERK2 (clone B3B9; Upstate Biotechnology); MEK1, MEK2, and paxillin (Transduction Labs); HA (clone 12CA5); myc (clone 9E10; Transduction Labs); p-FAK Y397 (Biosource International); PAK (anti-N20 for immunoprecipitation; anti-C19 for Western analysis); c-Raf (anti–C-12); and Gal4 transactivation domain (Santa Cruz Biotechnology, Inc.).

    Techniques: Staining

    Adhesion-dependent MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.

    Journal: The Journal of Cell Biology

    Article Title: PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation

    doi: 10.1083/jcb.200212141

    Figure Lengend Snippet: Adhesion-dependent MEK1 S298 phosphorylation promotes maximal MEK1 activation in response to growth factor stimulation. (A) REF52 cells were suspended for 90 min and either stimulated for 30 min with EGF and IGF-1 in suspension, replated on FN for 30 min, or stimulated with EGF and IGF-1 while they attached to FN for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (B) REF52 cells were placed in suspension for 90 min and either stimulated in suspension for 30 min with the indicated concentrations of EGF or replated on FN in the presence of the indicated concentrations of EGF for 30 min. Whole cell lysates were blotted with p-MAPK or ERK2 antisera. (C) Cells were treated as in A, and whole cell lysates were blotted with p-S218/222 MEK or MEK1 antisera. (D) REF52 cells were transiently transfected with HA-MEK1 (lanes 1–6) or HA-MEK1 S298A (lanes 7–12). Cells were suspended in serum-free media for 90 min and either kept unstimulated in suspension (lanes 1 and 7), stimulated in suspension with EGF for 20 min (lanes 2 and 8), allowed to adhere to FN (lanes 3 and 9) or allowed to attach to FN while stimulated with EGF for the indicated times (lanes 4–6 and 10–12). Anti-HA immunoprecipitates were formed and blotted with p-S218/222 MEK1, p-S298MEK1 or HA antisera. Densitometry and normalization to the loading controls revealed that S218/222 phosphorylation of MEK1 S298A was ∼50% that seen in the wild-type protein.

    Article Snippet: The following antibodies were used: phospho-MAPK (New England Biolabs, Inc.; ); MEK1/2 (New England Biolabs, Inc.); phospho-MEK1 S218/S222 (Sigma-Aldrich); ERK2 (clone B3B9; Upstate Biotechnology); MEK1, MEK2, and paxillin (Transduction Labs); HA (clone 12CA5); myc (clone 9E10; Transduction Labs); p-FAK Y397 (Biosource International); PAK (anti-N20 for immunoprecipitation; anti-C19 for Western analysis); c-Raf (anti–C-12); and Gal4 transactivation domain (Santa Cruz Biotechnology, Inc.).

    Techniques: Activation Assay, Transfection

    KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

    doi: 10.1128/MCB.01633-13

    Figure Lengend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

    Article Snippet: Western blot analysis was developed using the following primary and secondary antibodies (antibodies were from Cell Signaling unless otherwise noted): anti-p53 (Ab-7; Calbiochem) (1:2,500), anti-p19ARF (Abcam) (1:400), anti-p15INK4b (Biosource) (1:400), anti-MEK1/2 (1:1,000), anti-phospho-MEK1/2 (1:1,000), anti-phospho-ERK1/2 (1:1,000), anti-Ras (EMD-Biosciences) (1:1,000), anti-caveolin-1 (610059; BD Transduction Laboratories) (1:1000), anti-KSR1 (H-70; Santa Cruz) (1:500), anti-phospho-Ser338 c-Raf (1:500), anti-c-Raf (610151; BD Transduction Laboratories) (1:500), anti-B-Raf (H-145; Santa Cruz) (1:1,000), anti-peroxisome proliferator-activated receptor γ coactivator 1α (anti-PGC-1α) (H-300; Santa Cruz) (1:1,000), anti-estrogen-related receptor α (anti-ERRα) (V-19; Santa Cruz) (1:1,000), and anti-β-actin (Sigma) (1:3,000).

    Techniques: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence

    The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

    Journal: Molecular and Cellular Biology

    Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

    doi: 10.1128/MCB.01633-13

    Figure Lengend Snippet: The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

    Article Snippet: Western blot analysis was developed using the following primary and secondary antibodies (antibodies were from Cell Signaling unless otherwise noted): anti-p53 (Ab-7; Calbiochem) (1:2,500), anti-p19ARF (Abcam) (1:400), anti-p15INK4b (Biosource) (1:400), anti-MEK1/2 (1:1,000), anti-phospho-MEK1/2 (1:1,000), anti-phospho-ERK1/2 (1:1,000), anti-Ras (EMD-Biosciences) (1:1,000), anti-caveolin-1 (610059; BD Transduction Laboratories) (1:1000), anti-KSR1 (H-70; Santa Cruz) (1:500), anti-phospho-Ser338 c-Raf (1:500), anti-c-Raf (610151; BD Transduction Laboratories) (1:500), anti-B-Raf (H-145; Santa Cruz) (1:1,000), anti-peroxisome proliferator-activated receptor γ coactivator 1α (anti-PGC-1α) (H-300; Santa Cruz) (1:1,000), anti-estrogen-related receptor α (anti-ERRα) (V-19; Santa Cruz) (1:1,000), and anti-β-actin (Sigma) (1:3,000).

    Techniques: Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Western Blot, In Situ

    A : NES-B3T cells have higher levels of MEK1 phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

    doi: 10.1152/ajpgi.90262.2008

    Figure Lengend Snippet: A : NES-B3T cells have higher levels of MEK1 phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in

    Article Snippet: After separation and transfer to nitrocellulose membranes, the membranes were incubated with primary antibodies to p53 and p21 (Oncogene, San Diego, CA), cytokeratins (CK) 13 and 4 (Novocastra, Newcastle upon Tyne, UK), phospho-MEK1/2 (serines 217/221), phospho-MEK1 (threonine 286), phospho-ERK1/2, or MKP-1 (Cell Signaling Technology).

    Techniques: Activity Assay

    Acid increases MEK1/2 phosphorylation at activating sites (serines 217/221) in both cell lines, but acid increases ERK1/2 phosphorylation only in NES-G2T cells. Total ERK1/2, total MEK1/2, and β-tubulin were used as loading controls.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

    doi: 10.1152/ajpgi.90262.2008

    Figure Lengend Snippet: Acid increases MEK1/2 phosphorylation at activating sites (serines 217/221) in both cell lines, but acid increases ERK1/2 phosphorylation only in NES-G2T cells. Total ERK1/2, total MEK1/2, and β-tubulin were used as loading controls.

    Article Snippet: After separation and transfer to nitrocellulose membranes, the membranes were incubated with primary antibodies to p53 and p21 (Oncogene, San Diego, CA), cytokeratins (CK) 13 and 4 (Novocastra, Newcastle upon Tyne, UK), phospho-MEK1/2 (serines 217/221), phospho-MEK1 (threonine 286), phospho-ERK1/2, or MKP-1 (Cell Signaling Technology).

    Techniques:

    Phosphorylation of MEK1 on threonine 286 in esophageal squamous epithelium from 2 representative patients with GERD with Barrett's esophagus and 2 representative patients with GERD without Barrett's esophagus. Note expression of the inhibitory form of

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

    doi: 10.1152/ajpgi.90262.2008

    Figure Lengend Snippet: Phosphorylation of MEK1 on threonine 286 in esophageal squamous epithelium from 2 representative patients with GERD with Barrett's esophagus and 2 representative patients with GERD without Barrett's esophagus. Note expression of the inhibitory form of

    Article Snippet: After separation and transfer to nitrocellulose membranes, the membranes were incubated with primary antibodies to p53 and p21 (Oncogene, San Diego, CA), cytokeratins (CK) 13 and 4 (Novocastra, Newcastle upon Tyne, UK), phospho-MEK1/2 (serines 217/221), phospho-MEK1 (threonine 286), phospho-ERK1/2, or MKP-1 (Cell Signaling Technology).

    Techniques: Expressing

    Acid perfusion induces MEK1/2 phosphorylation on serines 217/222 in esophageal squamous epithelium of patients with GERD with and without Barrett's esophagus in vivo. A : representative Western blot demonstrating MEK1/2 phosphorylation and total MEK1/2

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

    doi: 10.1152/ajpgi.90262.2008

    Figure Lengend Snippet: Acid perfusion induces MEK1/2 phosphorylation on serines 217/222 in esophageal squamous epithelium of patients with GERD with and without Barrett's esophagus in vivo. A : representative Western blot demonstrating MEK1/2 phosphorylation and total MEK1/2

    Article Snippet: After separation and transfer to nitrocellulose membranes, the membranes were incubated with primary antibodies to p53 and p21 (Oncogene, San Diego, CA), cytokeratins (CK) 13 and 4 (Novocastra, Newcastle upon Tyne, UK), phospho-MEK1/2 (serines 217/221), phospho-MEK1 (threonine 286), phospho-ERK1/2, or MKP-1 (Cell Signaling Technology).

    Techniques: In Vivo, Western Blot

    MEK1 regulates oxidative stress and mitochondrial membrane function in ER + breast cancer cells . (a through c) MEK1 downregulation blocked the prosurvival effects of IGF-1 and enhanced ROS and mitochondrial membrane depolarization in hormonally treated breast cancer cells. (a) Western blot shows effective RNAi targeting of MEK1, which was carried out for 48 hours before cells were treated with E2, E2 + 4-OHT, or E2 + 4-OHT + MIF for 24 hours. Protein was isolated from cells and analyzed for MEK1 expression with immunoblot analysis. (b, c) Cell populations with reduced MEK1 expression were analyzed at 6 and 72 hours for ROS and mitochondrial membrane depolarization, respectively. (d through f) MEK1 overexpression reduced ROS and mitochondrial membrane depolarization in hormonally treated breast cancer cells. Transient transfection of MEK1 cDNA (MEK1-GFP vector) increased MEK1 expression above levels seen in vector-only (pEGFP-1)-transfected control cells at 24 and 48 hours, as determined by immunoblot analysis (d) , and reduced ROS levels and mitochondrial membrane depolarization at 24 and 72 hours, respectively (e, f) . Values are expressed as mean ± SD ( n = 3). Significant differences are designated as follows: (a) Scrambled versus SiMEK, E2 (± IGF-1); (b) Scrambled versus SiMEK, E2 + 4-OHT (± IGF-1); (c) Scrambled versus SiMEK, E2 + MIF (± IGF-1); (d) Scrambled versus SiMEK, E2 + 4-OHT + MIF; (e) pEGFP-N1 versus MEK1-GFP, E2 + 4-OHT; (f) pEGFP-N1 versus MEK1-GFP, E2 + MIF; (g) pEGFP-N1, E2 + 4-OHT + MIF versus MEK1-GFP, E2 + 4-OHT + MIF. * P

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: MEK1 regulates oxidative stress and mitochondrial membrane function in ER + breast cancer cells . (a through c) MEK1 downregulation blocked the prosurvival effects of IGF-1 and enhanced ROS and mitochondrial membrane depolarization in hormonally treated breast cancer cells. (a) Western blot shows effective RNAi targeting of MEK1, which was carried out for 48 hours before cells were treated with E2, E2 + 4-OHT, or E2 + 4-OHT + MIF for 24 hours. Protein was isolated from cells and analyzed for MEK1 expression with immunoblot analysis. (b, c) Cell populations with reduced MEK1 expression were analyzed at 6 and 72 hours for ROS and mitochondrial membrane depolarization, respectively. (d through f) MEK1 overexpression reduced ROS and mitochondrial membrane depolarization in hormonally treated breast cancer cells. Transient transfection of MEK1 cDNA (MEK1-GFP vector) increased MEK1 expression above levels seen in vector-only (pEGFP-1)-transfected control cells at 24 and 48 hours, as determined by immunoblot analysis (d) , and reduced ROS levels and mitochondrial membrane depolarization at 24 and 72 hours, respectively (e, f) . Values are expressed as mean ± SD ( n = 3). Significant differences are designated as follows: (a) Scrambled versus SiMEK, E2 (± IGF-1); (b) Scrambled versus SiMEK, E2 + 4-OHT (± IGF-1); (c) Scrambled versus SiMEK, E2 + MIF (± IGF-1); (d) Scrambled versus SiMEK, E2 + 4-OHT + MIF; (e) pEGFP-N1 versus MEK1-GFP, E2 + 4-OHT; (f) pEGFP-N1 versus MEK1-GFP, E2 + MIF; (g) pEGFP-N1, E2 + 4-OHT + MIF versus MEK1-GFP, E2 + 4-OHT + MIF. * P

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: Western Blot, Isolation, Expressing, Over Expression, Transfection, Plasmid Preparation

    BimEL expression levels vary between ER + breast cancer cell models and correlate to apoptotic outcome in response to hormonal treatments and MEK1 blockade . (a, b) Cell-number determinations showed that IGF-1 stimulated T-47D cell growth via a MEK1-dependent proliferation pathway. T-47D cells were treated with the indicated hormones in the presence or absence of IGF-1 (20 ng/ml) plus and minus PD 98059 for 216 hours (a) or U0126 for 144 hours (b) . (c) Western blot showed BimEL levels relative to the levels of cle aved PARP in T-47D cells treated with hormones in the absence or presence of U0126 for 72 hours. The levels of ER and PR are provided for validation of the ER and PR status of T-47D and MCF-7 cells used in this study. (d) Western blot compared the levels of BimEL in MCF-7 versus T-47D cells treated with hormones plus or minus U0126 for 48 h. (e) ROS levels were determined for T-47D and MCF-7 cells treated with the indicated hormones in the presence or absence of IGF-1 plus or minus MEK1 blockade with U0126. (f) Western blot showed that treatment with MG132 caused an accumulation of phosphorylated Bim EL in MCF-7 cells, but not in T-47D cells. (a through f) As described in Materials and Methods, at the indicated times, cells were harvested and analyzed either for cell counts (a, b) , protein expression by SDS/PAGE and immunoblotting for BimEL, pBimEL, pro-caspase-3, pMAPK, and total MAPK or β-actin, which were used as loading controls (c, d) , or for ROS determination (e) .

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: BimEL expression levels vary between ER + breast cancer cell models and correlate to apoptotic outcome in response to hormonal treatments and MEK1 blockade . (a, b) Cell-number determinations showed that IGF-1 stimulated T-47D cell growth via a MEK1-dependent proliferation pathway. T-47D cells were treated with the indicated hormones in the presence or absence of IGF-1 (20 ng/ml) plus and minus PD 98059 for 216 hours (a) or U0126 for 144 hours (b) . (c) Western blot showed BimEL levels relative to the levels of cle aved PARP in T-47D cells treated with hormones in the absence or presence of U0126 for 72 hours. The levels of ER and PR are provided for validation of the ER and PR status of T-47D and MCF-7 cells used in this study. (d) Western blot compared the levels of BimEL in MCF-7 versus T-47D cells treated with hormones plus or minus U0126 for 48 h. (e) ROS levels were determined for T-47D and MCF-7 cells treated with the indicated hormones in the presence or absence of IGF-1 plus or minus MEK1 blockade with U0126. (f) Western blot showed that treatment with MG132 caused an accumulation of phosphorylated Bim EL in MCF-7 cells, but not in T-47D cells. (a through f) As described in Materials and Methods, at the indicated times, cells were harvested and analyzed either for cell counts (a, b) , protein expression by SDS/PAGE and immunoblotting for BimEL, pBimEL, pro-caspase-3, pMAPK, and total MAPK or β-actin, which were used as loading controls (c, d) , or for ROS determination (e) .

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: Expressing, Western Blot, SDS Page

    A schematic representation of the role of BimEL and induction of apoptosis in ER + breast cancer cells . This model is a summary of data showing that MEK1 blockade, in addition to hormonal treatment (antiestrogen or antiprogestin treatment) will activate Bim via dephosphorylation and induce an ROS-dependent apoptotic cell death in some ER + breast cancer cells, particularly if IGF-1-induced IGF-IR signaling is active.

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: A schematic representation of the role of BimEL and induction of apoptosis in ER + breast cancer cells . This model is a summary of data showing that MEK1 blockade, in addition to hormonal treatment (antiestrogen or antiprogestin treatment) will activate Bim via dephosphorylation and induce an ROS-dependent apoptotic cell death in some ER + breast cancer cells, particularly if IGF-1-induced IGF-IR signaling is active.

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: De-Phosphorylation Assay

    The IGF-1/MEK signaling axis blocks the cytotoxic action of 4-OHT and/or MIF-treatments of ER + breast cancer cells . (a) Graphic representation of Western blot data showing pMAPK1/2 levels in cells treated with hormones in the absence and presence of IGF-1. The pMAPK1/2 level in E2-treated cells were arbitrarily set to a value of 100%; total MAPK levels served as the loading control. (b) Effective and selective blockade of MAPK1/2 phosphorylation by the MEK1 inhibitor PD 98059. Total MAPK1/2 and AKT levels served as loading controls. (c, d) PD 98059 blocked the proliferative effects of IGF-1 and restored the ability of 4-OHT and/or MIF therapy to induce cell detachment and cleavage of PARP (apoptosis) in the MCF-7 cell populations treated with 4-OHT and/or MIF. fold diff ., levels of cleaved PARP after correction for differences in protein loading; β- actin levels served as loading controls. (a-d) Cells were treated for the indicated time periods with hormones, as indicated in the absence or presence of IGF-1 at 20 ng/ml and/or PD 98059 at 25 μ;g/ml and harvested for immunoblotting or cell counts, as described in Materials and Methods. Values are expressed as mean ± SD ( n = 3). Treatment effects on total cell number were determined to be significant when compared with (a) E2; (b) E2 + IGF-1; (c) E2 + 4-OHT + IGF-1; (d) E2 + MIF + IGF-1; (e) E2 + IGF-1 + PD 98059. * P

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: The IGF-1/MEK signaling axis blocks the cytotoxic action of 4-OHT and/or MIF-treatments of ER + breast cancer cells . (a) Graphic representation of Western blot data showing pMAPK1/2 levels in cells treated with hormones in the absence and presence of IGF-1. The pMAPK1/2 level in E2-treated cells were arbitrarily set to a value of 100%; total MAPK levels served as the loading control. (b) Effective and selective blockade of MAPK1/2 phosphorylation by the MEK1 inhibitor PD 98059. Total MAPK1/2 and AKT levels served as loading controls. (c, d) PD 98059 blocked the proliferative effects of IGF-1 and restored the ability of 4-OHT and/or MIF therapy to induce cell detachment and cleavage of PARP (apoptosis) in the MCF-7 cell populations treated with 4-OHT and/or MIF. fold diff ., levels of cleaved PARP after correction for differences in protein loading; β- actin levels served as loading controls. (a-d) Cells were treated for the indicated time periods with hormones, as indicated in the absence or presence of IGF-1 at 20 ng/ml and/or PD 98059 at 25 μ;g/ml and harvested for immunoblotting or cell counts, as described in Materials and Methods. Values are expressed as mean ± SD ( n = 3). Treatment effects on total cell number were determined to be significant when compared with (a) E2; (b) E2 + IGF-1; (c) E2 + 4-OHT + IGF-1; (d) E2 + MIF + IGF-1; (e) E2 + IGF-1 + PD 98059. * P

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: Western Blot

    SX13 cells expressing low-levels of IGF-1R are sensitive to the death-inducing effects of PD 98059 . (a) Western blot showing low levels of IGF-1R, but comparable levels of ERα and cleaved PARP in SX13 and NEO cells undergoing the indicated hormonal treatments for 120 hours. (b, c) Cell counts showing that IGF-1 does not enhance E2-stimulated SX13 cell proliferation, but that PD 98059 can restore the growth-inhibitory effects of 4-OHT treatment. Cells (2 × 10 5 ) were seeded and, after 24 hours, treated with either 1% or 5% FBS-DCC serum in the absence (E2 ablation) or presence of E2 and/or IGF-1 for 168 hours (b) or 144 hours (c) . Cell counts were performed with a Coulter counter (c) . (d) Trypan blue exclusion assay shows that IGF-1 attenuates the death-inducing effects of 4-OHT and/or MIF treatments in an MEK1-dependent manner. At 144 hours after treatments, adherent and detached cells were collected and counted by using a hemacytometer. (e) Representative images show that PD 98059 effectively reduces cell number in the E2-treated cell population (compare a with b ), and induces cell shrinkage and detachment, indicative of apoptosis, in the 4-OHT plus MIF-treated cell population (compare c with d ). Data in ( b through d ) are expressed as mean ± SD ( n = 3). The following show significant differences in the induction of apoptosis (number of detached cells) and cell proliferation for the hormonal therapies compared with: (a) E2; (b) 4-OHT + MIF; (c) E2+IGF-1; (d) E2+IGF-1+PD 98059. * P

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: SX13 cells expressing low-levels of IGF-1R are sensitive to the death-inducing effects of PD 98059 . (a) Western blot showing low levels of IGF-1R, but comparable levels of ERα and cleaved PARP in SX13 and NEO cells undergoing the indicated hormonal treatments for 120 hours. (b, c) Cell counts showing that IGF-1 does not enhance E2-stimulated SX13 cell proliferation, but that PD 98059 can restore the growth-inhibitory effects of 4-OHT treatment. Cells (2 × 10 5 ) were seeded and, after 24 hours, treated with either 1% or 5% FBS-DCC serum in the absence (E2 ablation) or presence of E2 and/or IGF-1 for 168 hours (b) or 144 hours (c) . Cell counts were performed with a Coulter counter (c) . (d) Trypan blue exclusion assay shows that IGF-1 attenuates the death-inducing effects of 4-OHT and/or MIF treatments in an MEK1-dependent manner. At 144 hours after treatments, adherent and detached cells were collected and counted by using a hemacytometer. (e) Representative images show that PD 98059 effectively reduces cell number in the E2-treated cell population (compare a with b ), and induces cell shrinkage and detachment, indicative of apoptosis, in the 4-OHT plus MIF-treated cell population (compare c with d ). Data in ( b through d ) are expressed as mean ± SD ( n = 3). The following show significant differences in the induction of apoptosis (number of detached cells) and cell proliferation for the hormonal therapies compared with: (a) E2; (b) 4-OHT + MIF; (c) E2+IGF-1; (d) E2+IGF-1+PD 98059. * P

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: Expressing, Western Blot, Droplet Countercurrent Chromatography, Trypan Blue Exclusion Assay

    Blockade of MEK1 increases the levels of dephosphorylated BimEL in MCF-7 . (a through c) Western blot shows the expression level of the Bim isoforms (EL, extra long; L, long, and/or S, short) in cells treated with hormones plus and minus IGF-1 (a) , with two apparent size variants of BimEL, a high-molecular-weight BimEL (top band, denoted by the dash) and a low-molecular-weight BimEL (bottom band, denoted by the arrow) (b, c) . fold diff ., fold difference in signal intensity of the lower-molecular-weight BimEL band divided by the signal intensity of the high-molecular-weight BimEL band by using the total MAPK signal intensity per lane as the loading control. (d, e) Western blot shows the accumulation of the upper Bim EL band when cells with active MEK1 are treated with the proteasome inhibitor MG132, and preferential loss of the upper BimEL band, concomitant with accumulation of the lower-molecular-weight Bim EL form after λ protein phosphatase treatment of the protein lysates. The λ protein phosphatase digestion (described in Materials and Methods) was carried out for 20 minutes (lanes 2 and 5) or 1 hour (lane 3) on protein lysates isolated from breast cancer cells undergoing E2 treatment for 24 hours. Immunoblotting determined the levels of BimEL and pMAPK1/2; total MAPK served as loading control. (f) Western blot shows that TAM- and/or U0126- treated cells show significantly higher levels of BimEL protein than do E2-treated cells, with the highest levels of dephosphorylated Bim EL, correlating directly to the cleavage of PARP detectable by 72 hours.

    Journal: Breast Cancer Research : BCR

    Article Title: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

    doi: 10.1186/bcr3153

    Figure Lengend Snippet: Blockade of MEK1 increases the levels of dephosphorylated BimEL in MCF-7 . (a through c) Western blot shows the expression level of the Bim isoforms (EL, extra long; L, long, and/or S, short) in cells treated with hormones plus and minus IGF-1 (a) , with two apparent size variants of BimEL, a high-molecular-weight BimEL (top band, denoted by the dash) and a low-molecular-weight BimEL (bottom band, denoted by the arrow) (b, c) . fold diff ., fold difference in signal intensity of the lower-molecular-weight BimEL band divided by the signal intensity of the high-molecular-weight BimEL band by using the total MAPK signal intensity per lane as the loading control. (d, e) Western blot shows the accumulation of the upper Bim EL band when cells with active MEK1 are treated with the proteasome inhibitor MG132, and preferential loss of the upper BimEL band, concomitant with accumulation of the lower-molecular-weight Bim EL form after λ protein phosphatase treatment of the protein lysates. The λ protein phosphatase digestion (described in Materials and Methods) was carried out for 20 minutes (lanes 2 and 5) or 1 hour (lane 3) on protein lysates isolated from breast cancer cells undergoing E2 treatment for 24 hours. Immunoblotting determined the levels of BimEL and pMAPK1/2; total MAPK served as loading control. (f) Western blot shows that TAM- and/or U0126- treated cells show significantly higher levels of BimEL protein than do E2-treated cells, with the highest levels of dephosphorylated Bim EL, correlating directly to the cleavage of PARP detectable by 72 hours.

    Article Snippet: Immunoblotting was conducted according to the manufacturer's protocol by using primary antibodies to: LC3 (ab48394), p62 (ab56416), cleaved lamin A (ab52300) [Abcam]; cleaved PARP (9541), phospho-p44/42 MAP kinase (Thr202/Tyr204, 9106), total MAPK (9102), Akt (9272), phospho-Akt (Ser473, 9271), MEK1 (9124), pBim (4581), and Bim (2819) [Cell Signaling]; pERK1/2 (SC-7383), ERα (SC-8002), and IGF-1Rβ (SC-713) [Santa Cruz Biotechnology]; and β-actin (A5441) [Sigma].

    Techniques: Western Blot, Expressing, Molecular Weight, Isolation

    MEK1 is required to inhibit the expression of the pluripotency genes pou5f3.2 and ventx2 . ( A ) Embryos injected with 25 ng Mk-MO at 16-cell stage in one animal dorsal blastomere were grown until late gastrulation stage 13 and processed for WISH with pou5f3.2 and ventx2 probes. ( B ) Embryos injected with 25 ng Mk-MO at 16 cell stage in one animal ventral blastomere were grown until mid-neurula stage 18 and processed for WISH with pou5f3.2 probe. ( C ) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO and grown until blastula stage 9, when animal caps were isolated, cultured in vitro until late gastrula stage 13 and then processed for RT-qPCR. In A and B, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. DOI: http://dx.doi.org/10.7554/eLife.21526.008

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: MEK1 is required to inhibit the expression of the pluripotency genes pou5f3.2 and ventx2 . ( A ) Embryos injected with 25 ng Mk-MO at 16-cell stage in one animal dorsal blastomere were grown until late gastrulation stage 13 and processed for WISH with pou5f3.2 and ventx2 probes. ( B ) Embryos injected with 25 ng Mk-MO at 16 cell stage in one animal ventral blastomere were grown until mid-neurula stage 18 and processed for WISH with pou5f3.2 probe. ( C ) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO and grown until blastula stage 9, when animal caps were isolated, cultured in vitro until late gastrula stage 13 and then processed for RT-qPCR. In A and B, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. DOI: http://dx.doi.org/10.7554/eLife.21526.008

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: Expressing, Injection, Isolation, Cell Culture, In Vitro, Quantitative RT-PCR

    Neural induction in vivo depends on MEK1 activity. Sixteen-cell embryos were injected in one ventral-animal blastomere with 3 ng of dominant-negative Smad5 (Smad5sbn) mRNA and 25 ng Mk-MO ATG, as indicated. Embryos were fixed at late gastrula stage 13, and processed for WISH with the indicated probes. The number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. Lateral views. DOI: http://dx.doi.org/10.7554/eLife.21526.007

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: Neural induction in vivo depends on MEK1 activity. Sixteen-cell embryos were injected in one ventral-animal blastomere with 3 ng of dominant-negative Smad5 (Smad5sbn) mRNA and 25 ng Mk-MO ATG, as indicated. Embryos were fixed at late gastrula stage 13, and processed for WISH with the indicated probes. The number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. Lateral views. DOI: http://dx.doi.org/10.7554/eLife.21526.007

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: In Vivo, Activity Assay, Injection, Dominant Negative Mutation

    Ventx2 degradation and asymmetric distribution require MEK1 activity. ( A ) In silico analysis of phosphorylation sites in the Ventx2 protein and prediction of kinases involved, with Kinasephos2 software. ( B ) Schematic representation of the Ventx2 protein. HD indicates the homeodomain (blue box), and the PEST destruction motif is highlighted in red. Note that Serine 140, which is required for Ventx2 degradation, is a predicted target of MAPK. ( C ) 50 pg Ventx2-Myc RNA was injected into both blastomeres at the two-cell stage. 50 pg GFP-Myc-RNA was co-injected as an internal loading control. Embryos were allowed to develop until the indicated stages, and exogenous Ventx2 was detected by anti-Myc immunostaining on Western blot. The graph shows the ratios of Ventx2-Myc over a-tub signals measured from the Western blot. ( D ) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs and with 25 ng Mk-MO as indicated. Embryos were fixed at blastula stage 9, cryosectioned and processed for anti-Myc (red), anti-GFP (green) and anti-g-tubulin (centrosomes, white) immunostaining, and DNA was stained with DAPI (blue). Panels represent compiled confocal slices to visualize entire mitotic nuclei. DOI: http://dx.doi.org/10.7554/eLife.21526.011

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: Ventx2 degradation and asymmetric distribution require MEK1 activity. ( A ) In silico analysis of phosphorylation sites in the Ventx2 protein and prediction of kinases involved, with Kinasephos2 software. ( B ) Schematic representation of the Ventx2 protein. HD indicates the homeodomain (blue box), and the PEST destruction motif is highlighted in red. Note that Serine 140, which is required for Ventx2 degradation, is a predicted target of MAPK. ( C ) 50 pg Ventx2-Myc RNA was injected into both blastomeres at the two-cell stage. 50 pg GFP-Myc-RNA was co-injected as an internal loading control. Embryos were allowed to develop until the indicated stages, and exogenous Ventx2 was detected by anti-Myc immunostaining on Western blot. The graph shows the ratios of Ventx2-Myc over a-tub signals measured from the Western blot. ( D ) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs and with 25 ng Mk-MO as indicated. Embryos were fixed at blastula stage 9, cryosectioned and processed for anti-Myc (red), anti-GFP (green) and anti-g-tubulin (centrosomes, white) immunostaining, and DNA was stained with DAPI (blue). Panels represent compiled confocal slices to visualize entire mitotic nuclei. DOI: http://dx.doi.org/10.7554/eLife.21526.011

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: Activity Assay, In Silico, Software, Injection, Immunostaining, Western Blot, Staining

    Ventx2 knockdown restores germ-layer formation in MEK1-deficient embryos. ( A ) Four-cell embryos were injected with 50 pg 2SAVentx2-Myc RNA per cell, fixed at tailbud stage 25 and processed for WISH with pou5f3.2 probe. ( B ) Four-cell embryos were injected with 30 ng Ventx2-MO (Vx2-MO) per blastomere, collected at stage 10.5 and processed for RT-qPCR. ( C ) Embryos injected as in B were processed for WISH analysis at early gastrula stage 10.5 with ventx1 and gsc probes (vegetal view). ( D ). Four-cell embryos were injected with 25 ng Mk-MO, with or without 7.5 ng Vx2-MO, in each blastomere, collected at gastrula stage 10.5, and processed for WISH with indicated probes. Note that embryos stained for xk81a1 (epidermis) were injected in one ventral animal blastomere at 16 cell stage and collected at late gastrula stage 13. Embryos stained for gsc were hemisectioned prior to staining to improve probe penetration. In A and D, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. For the RT-qPCR graph, error bars represent s.e.m. values of three independent experiments with two technical duplicates. For statistical analysis, samples from injected embryos were compared with samples from uninjected control embryos by Unpaired Student’s t-test. *p

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: Ventx2 knockdown restores germ-layer formation in MEK1-deficient embryos. ( A ) Four-cell embryos were injected with 50 pg 2SAVentx2-Myc RNA per cell, fixed at tailbud stage 25 and processed for WISH with pou5f3.2 probe. ( B ) Four-cell embryos were injected with 30 ng Ventx2-MO (Vx2-MO) per blastomere, collected at stage 10.5 and processed for RT-qPCR. ( C ) Embryos injected as in B were processed for WISH analysis at early gastrula stage 10.5 with ventx1 and gsc probes (vegetal view). ( D ). Four-cell embryos were injected with 25 ng Mk-MO, with or without 7.5 ng Vx2-MO, in each blastomere, collected at gastrula stage 10.5, and processed for WISH with indicated probes. Note that embryos stained for xk81a1 (epidermis) were injected in one ventral animal blastomere at 16 cell stage and collected at late gastrula stage 13. Embryos stained for gsc were hemisectioned prior to staining to improve probe penetration. In A and D, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. For the RT-qPCR graph, error bars represent s.e.m. values of three independent experiments with two technical duplicates. For statistical analysis, samples from injected embryos were compared with samples from uninjected control embryos by Unpaired Student’s t-test. *p

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: Injection, Quantitative RT-PCR, Staining

    MEK1 depletion by morpholinos. ( A ) Four-cell embryos were injected in each blastomere with 50 pg GFP‐CAAX mRNA with or without 25 ng Mk-MO, fixed at blastula stage 9, cryosectioned and stained with anti-phospho-MEK1 antibody. The pMEK1 signal was severely reduced or lost in cells injected with Mk-MO. ( B ) Four-cell embryos were injected in each cell with 25 ng Mk-MO, collected at early gastrula stage 10.5 and processed for RT-qPCR to quantify p53 expression levels. Mk‐MO did not induce p53 expression. ( C ) Two-cell embryos were injected twice with 25 ng Mk-MO in each blastomere, followed by injection at 4 cell stage of hamster MEK1 mRNA (Mk; 400 pg per blastomere). In order to score progress through gastrulation pictures were taken from live stage 13 gastrula embryos (vegetal view). ( D ) Blastopore closure was scored by calculating the ratio of blastopore diameter of injected embryos to the mean of blastopore diameter of uninjected control embryos. Bars represent maximum and minimum values, and lines represent the mean. The number of embryos analyzed in each condition is displayed on the graph. For statistical analysis, samples were compared by Mann-Whitney test (99% confidence intervals were applied; ***p≤0.0001). DOI: http://dx.doi.org/10.7554/eLife.21526.003 10.7554/eLife.21526.004 Values of blastopore closure ratios. Details are shown in Figure 1—figure supplement 1 and Materials and methods. DOI: http://dx.doi.org/10.7554/eLife.21526.004

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: MEK1 depletion by morpholinos. ( A ) Four-cell embryos were injected in each blastomere with 50 pg GFP‐CAAX mRNA with or without 25 ng Mk-MO, fixed at blastula stage 9, cryosectioned and stained with anti-phospho-MEK1 antibody. The pMEK1 signal was severely reduced or lost in cells injected with Mk-MO. ( B ) Four-cell embryos were injected in each cell with 25 ng Mk-MO, collected at early gastrula stage 10.5 and processed for RT-qPCR to quantify p53 expression levels. Mk‐MO did not induce p53 expression. ( C ) Two-cell embryos were injected twice with 25 ng Mk-MO in each blastomere, followed by injection at 4 cell stage of hamster MEK1 mRNA (Mk; 400 pg per blastomere). In order to score progress through gastrulation pictures were taken from live stage 13 gastrula embryos (vegetal view). ( D ) Blastopore closure was scored by calculating the ratio of blastopore diameter of injected embryos to the mean of blastopore diameter of uninjected control embryos. Bars represent maximum and minimum values, and lines represent the mean. The number of embryos analyzed in each condition is displayed on the graph. For statistical analysis, samples were compared by Mann-Whitney test (99% confidence intervals were applied; ***p≤0.0001). DOI: http://dx.doi.org/10.7554/eLife.21526.003 10.7554/eLife.21526.004 Values of blastopore closure ratios. Details are shown in Figure 1—figure supplement 1 and Materials and methods. DOI: http://dx.doi.org/10.7554/eLife.21526.004

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: Injection, Staining, Quantitative RT-PCR, Expressing, MANN-WHITNEY

    Gene expression analysis of MEK1-depleted gastrula embryos. ( A–D ) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO, collected at early gastrula stage 10.5 and processed for RT-qPCR to quantify changes in the expression levels of pro-differentiation markers ( A ), or changes in the expression levels of BMPs (Smad1/5/8) and Activin/Nodal (Smad2/3) signaling targets ( C ). For all qPCR graphs, error bars represent s.e.m. values of four independent experiments with two technical duplicates. For statistical analyses, samples from injected embryos were compared with samples from uninjected control embryos by Unpaired Student’s t-test. *p

    Journal: eLife

    Article Title: Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2

    doi: 10.7554/eLife.21526

    Figure Lengend Snippet: Gene expression analysis of MEK1-depleted gastrula embryos. ( A–D ) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO, collected at early gastrula stage 10.5 and processed for RT-qPCR to quantify changes in the expression levels of pro-differentiation markers ( A ), or changes in the expression levels of BMPs (Smad1/5/8) and Activin/Nodal (Smad2/3) signaling targets ( C ). For all qPCR graphs, error bars represent s.e.m. values of four independent experiments with two technical duplicates. For statistical analyses, samples from injected embryos were compared with samples from uninjected control embryos by Unpaired Student’s t-test. *p

    Article Snippet: Primary antibodies were as follows: anti-Myc (9E10; Santa Cruz Biotech, dilution 1/300 RRID: AB_627268 ), anti-GFP (GFP-1020; 2BScientific; dilution 1/1000 RRID: AB_10000240 ), anti-γ-Tubulin (ab16504; Abcam; dilution 1/1000 RRID: AB_443396 ), anti-phospho-MEK1 (9121; Cell Signaling Technology; dilution 1/400 RRID: AB_331648 ).

    Techniques: Expressing, Injection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Ephrin-B1 stimulation of EphB2 leads to the down regulation of the ERK1/2 MAPK signaling pathway. (A and B) Serum-starved (right panels) or growing (10% FBS) (left panels) parental NG108 or NG-EphB2 cells were stimulated with 2 μg of clustered Fc-ephrin-B1 per ml for the indicated time points and lysed directly in 2x SDS-PAGE sample buffer. The lysates were electrophoresed and blotted (WB) with antibodies against phosphorylated ERK1/2 (A, top panels), or phosphorylated MEK1 (B, top panels). The blots were stripped and reprobed for total ERK1 or MEK1 (bottom panels, A and B, respectively). (C) Graphical representation of phospho-ERK1/2 and phospho -MEK1 from panels A and B relative to total levels. (D) Time course of EphB2 tyrosine phosphorylation in NG108 cells. NG108 cells were serum starved (lanes −serum) or grown in the presence of serum (lanes +serum) and stimulated with 2 μg of clustered Fc-ephrin-B1 per ml as indicated. Immunoprecipitated (IP) EphB2 was then resolved by SDS-PAGE and probed with anti-pTyr antibodies (upper panel). Blots were subsequently stripped and reprobed with crude anti-EphB2 sera (lower panel).

    Journal: Molecular and Cellular Biology

    Article Title: Downregulation of the Ras-Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    doi: 10.1128/MCB.21.21.7429-7441.2001

    Figure Lengend Snippet: Ephrin-B1 stimulation of EphB2 leads to the down regulation of the ERK1/2 MAPK signaling pathway. (A and B) Serum-starved (right panels) or growing (10% FBS) (left panels) parental NG108 or NG-EphB2 cells were stimulated with 2 μg of clustered Fc-ephrin-B1 per ml for the indicated time points and lysed directly in 2x SDS-PAGE sample buffer. The lysates were electrophoresed and blotted (WB) with antibodies against phosphorylated ERK1/2 (A, top panels), or phosphorylated MEK1 (B, top panels). The blots were stripped and reprobed for total ERK1 or MEK1 (bottom panels, A and B, respectively). (C) Graphical representation of phospho-ERK1/2 and phospho -MEK1 from panels A and B relative to total levels. (D) Time course of EphB2 tyrosine phosphorylation in NG108 cells. NG108 cells were serum starved (lanes −serum) or grown in the presence of serum (lanes +serum) and stimulated with 2 μg of clustered Fc-ephrin-B1 per ml as indicated. Immunoprecipitated (IP) EphB2 was then resolved by SDS-PAGE and probed with anti-pTyr antibodies (upper panel). Blots were subsequently stripped and reprobed with crude anti-EphB2 sera (lower panel).

    Article Snippet: Phospho-ERK1/2 and phospho-MEK1 antibodies were purchased from Cell Signaling Technologies, and Grb2 antibodies were purchased from Transduction Laboratories.

    Techniques: SDS Page, Western Blot, Immunoprecipitation

    Kinase activity of EphB2 is required for down regulation of ERK1/2 signaling. (A) Schematic representation of the EphB2 mutant structures, indicating juxtamembrane tyrosines JX1 (Y 604 ), JX2 (Y 610 ) and the conserved SAM domain tyrosine (Y 929 ). (B and C) EphB2-ΔC, a C-terminal truncation, ends at V 951 in the SAM domain. NG108 or NG-EphB2 clones expressing WT or mutant receptors were stimulated with 2 μg of clustered Fc-ephrin-B1 per ml for the indicated time points. The cells were harvested directly in 2× SDS sample buffer, and lysates were electrophoresed and immunoblotted (WB) with antibodies against phosphorylated ERK1/2 (B, top panel) or phosphorylated MEK1 (C, top panel). Immunoblots were stripped and reprobed with anti-ERK1 (B, bottom panel) or anti-MEK1 (C, bottom panel). (D) NG108 cells expressing WT EphB2 and kinase-inactive EphB2 (KD) were stimulated with clustered Fc-ephrin-B1 and treated as indicated for panels B and C. Lysates were then probed with anti-phospho-ERK1/2 (upper panel), and anti-ERK1 (lower panel).

    Journal: Molecular and Cellular Biology

    Article Title: Downregulation of the Ras-Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    doi: 10.1128/MCB.21.21.7429-7441.2001

    Figure Lengend Snippet: Kinase activity of EphB2 is required for down regulation of ERK1/2 signaling. (A) Schematic representation of the EphB2 mutant structures, indicating juxtamembrane tyrosines JX1 (Y 604 ), JX2 (Y 610 ) and the conserved SAM domain tyrosine (Y 929 ). (B and C) EphB2-ΔC, a C-terminal truncation, ends at V 951 in the SAM domain. NG108 or NG-EphB2 clones expressing WT or mutant receptors were stimulated with 2 μg of clustered Fc-ephrin-B1 per ml for the indicated time points. The cells were harvested directly in 2× SDS sample buffer, and lysates were electrophoresed and immunoblotted (WB) with antibodies against phosphorylated ERK1/2 (B, top panel) or phosphorylated MEK1 (C, top panel). Immunoblots were stripped and reprobed with anti-ERK1 (B, bottom panel) or anti-MEK1 (C, bottom panel). (D) NG108 cells expressing WT EphB2 and kinase-inactive EphB2 (KD) were stimulated with clustered Fc-ephrin-B1 and treated as indicated for panels B and C. Lysates were then probed with anti-phospho-ERK1/2 (upper panel), and anti-ERK1 (lower panel).

    Article Snippet: Phospho-ERK1/2 and phospho-MEK1 antibodies were purchased from Cell Signaling Technologies, and Grb2 antibodies were purchased from Transduction Laboratories.

    Techniques: Activity Assay, Mutagenesis, Clone Assay, Expressing, Western Blot

    PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1 −/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1 −/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1 −/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and phospho-Ser298-MEK1/2 levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×10 5 Wt or PAK1 −/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.

    Journal: Journal of cell science

    Article Title: PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics

    doi: 10.1242/jcs.027680

    Figure Lengend Snippet: PAK1 regulates CSF-1-induced MAPK activation but not macrophage differentiation or chemotaxis. A, lysates from Wt and PAK1 −/− BMMs were immunoblotted for PAK1 and PAK2 using a group 1 specific polyclonal antibody (C19) or a PAK1-specific polyclonal antibody. β-actin was used as a loading control. B, Flow cytometry analysis of Wt and PAK1 −/− BMMs surface F4/80 expression levels, detected using a FITC-F4/80 antibody. Background fluorescence levels were established with a FITC-IgG2b negative control antibody. C, Wt BMMs were stimulated with 33 ng/ml CSF-1, and lysates were immunoblotted for phospho-Thr423-PAK1 and β-actin as a loading control. D, Wt and PAK1 −/− BMMs were stimulated with 33 ng/ml CSF-1 and lysates were immunoblotted for phospho-Ser473-Akt, phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38 and phospho-Ser298-MEK1/2 levels. β-actin was used as a loading control. Western blots are representative of three separate experiments. E, To investigate chemotaxis, 1×10 5 Wt or PAK1 −/− BMMs were placed into the upper chamber of a Transwell with 33 ng/ml CSF-1 in the lower chamber. After 24 hours, cell migration was evaluated by determining the cell number in ten randomly selected fields. Results are the mean +/− s.e.m. of 3 experiments performed in triplicate.

    Article Snippet: Membranes were then incubated at 4°C for 16 hours in 0.5% non-fat dried milk with rabbit polyclonal anti-PAK1, 2 and 3 (C19, Santa Cruz), anti-PAK1, anti-phospho-Thr423-PAK, anti-phospho-Ser473-Akt, anti-phospho-Thr202/Tyr204-ERK1/2, anti-ERK1/2, anti-phospho-Ser298-MEK1, anti-phospho-S217/221-MEK1, anti-phospho-Ser338-c-Raf (all from Cell Signalling Technology), anti-phospho-Thr180/Tyr182-p38 antibodies (New England Biolabs), anti-Rac1 (Upstate Biotechnology) or mouse monoclonal anti-β actin (Sigma).

    Techniques: Activation Assay, Chemotaxis Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Negative Control, Western Blot, Migration

    PAK1 promotes ERK1/2 activation at the cell periphery. A, Wt and PAK1 −/− BMMs were adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38, phospho-Ser298-MEK1/2 and total ERK1/2. Densitometry quantification of phosphorylated ERK1/2 and p38 levels equalised to total ERK1/2 protein levels are shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments. B, Wt and PAK1 −/− BMMs were plated onto glass coverslips for 10 minutes in growth medium and were stained using an ERK1/2 antibody and TRITC-phalloidin to visualise F-actin. Cells were imaged by confocal microscopy. ERK1/2 localisation was quantified by determining the number of cells with ERK1/2 staining at the periphery. The mean +/− s.d. is shown for two separate experiments, n = 60 (Wt) and 45 (PAK1 −/− ). C, BMMs were stained with a phospho-Thr202/Tyr204-ERK1/2 antibody (P-ERK1/2) and TRITC-phalloidin (F-actin). Localisation of phospho-ERK1/2 was quantified by determining the number of cells with staining at the cell periphery. The mean +/− s.d. is shown for two separate experiments, n = 39 (Wt) and 62 (PAK1 −/− ). D, Wt and PAK1−/− BMMs were kept in suspension or adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Ser217/221-MEK1/2, and total ERK1/2 as a loading control. Densitometry quantification of phosphorylated MEK1/2 equalised to total ERK1/2 protein levels is shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments.

    Journal: Journal of cell science

    Article Title: PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics

    doi: 10.1242/jcs.027680

    Figure Lengend Snippet: PAK1 promotes ERK1/2 activation at the cell periphery. A, Wt and PAK1 −/− BMMs were adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Thr202/Tyr204-ERK1/2, phospho-Thr180/Tyr182-p38, phospho-Ser298-MEK1/2 and total ERK1/2. Densitometry quantification of phosphorylated ERK1/2 and p38 levels equalised to total ERK1/2 protein levels are shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments. B, Wt and PAK1 −/− BMMs were plated onto glass coverslips for 10 minutes in growth medium and were stained using an ERK1/2 antibody and TRITC-phalloidin to visualise F-actin. Cells were imaged by confocal microscopy. ERK1/2 localisation was quantified by determining the number of cells with ERK1/2 staining at the periphery. The mean +/− s.d. is shown for two separate experiments, n = 60 (Wt) and 45 (PAK1 −/− ). C, BMMs were stained with a phospho-Thr202/Tyr204-ERK1/2 antibody (P-ERK1/2) and TRITC-phalloidin (F-actin). Localisation of phospho-ERK1/2 was quantified by determining the number of cells with staining at the cell periphery. The mean +/− s.d. is shown for two separate experiments, n = 39 (Wt) and 62 (PAK1 −/− ). D, Wt and PAK1−/− BMMs were kept in suspension or adhered onto tissue culture plastic for 10 minutes in growth medium. Lysates were immunoblotted for phospho-Ser217/221-MEK1/2, and total ERK1/2 as a loading control. Densitometry quantification of phosphorylated MEK1/2 equalised to total ERK1/2 protein levels is shown (a.u., arbitrary units). Data show the mean +/− s.d. of two separate experiments.

    Article Snippet: Membranes were then incubated at 4°C for 16 hours in 0.5% non-fat dried milk with rabbit polyclonal anti-PAK1, 2 and 3 (C19, Santa Cruz), anti-PAK1, anti-phospho-Thr423-PAK, anti-phospho-Ser473-Akt, anti-phospho-Thr202/Tyr204-ERK1/2, anti-ERK1/2, anti-phospho-Ser298-MEK1, anti-phospho-S217/221-MEK1, anti-phospho-Ser338-c-Raf (all from Cell Signalling Technology), anti-phospho-Thr180/Tyr182-p38 antibodies (New England Biolabs), anti-Rac1 (Upstate Biotechnology) or mouse monoclonal anti-β actin (Sigma).

    Techniques: Activation Assay, Staining, Confocal Microscopy

    DA-Raf-dependent inactivation of MEK1/2 suppresses TIMP4 expression and induces a subsequent proteolytic cascade MMP14–MMP2. ( A ) Total lungs and AEC2s were collected from X/Y (pink boxes) and X – /Y (blue boxes) mice at P5, and the expression levels of Timp4 were analyzed by using real-time PCR. ( B ) Lung lysates isolated from X/Y and X – /Y mice were subjected to the gelatin zymography analysis. ( C ) Activity levels of MMP14 in the lungs of X/Y (pink bar) and X – . The values represent means ± SD. ( D ) The expression levels of Mmp2 , Mmp9 , and Mmp14 in lungs at P5 from X/Y and X – /Y mice were analyzed by real-time PCR. Values represent the amounts of mRNA relative to those in WT littermates, which are arbitrarily defined as 1. ( E ) MMP2 activity ( Top ) was analyzed by zymography using lysates from developing lungs. The amounts of TIMP4 ( Middle ) and β-Tubulin ( Bottom ) were examined by Western blot analysis at indicated postnatal ages. ( F and G ) The expression levels of Timp4 in lungs at P6 from X/Y and X – /Y mice treated with DMSO or MEKi were examined by using real-time PCR ( F ) or Western blot analysis ( G ). ( H ) MMP2 activity levels were analyzed gelatin zymography by use of lung lysates isolated from X/Y and X – /Y mice treated with DMSO or the MEKi. In A , C , D , and F the analyses were performed with three to five independent mice per genotype. ** P

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

    Article Title: DA-Raf–dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation

    doi: 10.1073/pnas.1321574111

    Figure Lengend Snippet: DA-Raf-dependent inactivation of MEK1/2 suppresses TIMP4 expression and induces a subsequent proteolytic cascade MMP14–MMP2. ( A ) Total lungs and AEC2s were collected from X/Y (pink boxes) and X – /Y (blue boxes) mice at P5, and the expression levels of Timp4 were analyzed by using real-time PCR. ( B ) Lung lysates isolated from X/Y and X – /Y mice were subjected to the gelatin zymography analysis. ( C ) Activity levels of MMP14 in the lungs of X/Y (pink bar) and X – . The values represent means ± SD. ( D ) The expression levels of Mmp2 , Mmp9 , and Mmp14 in lungs at P5 from X/Y and X – /Y mice were analyzed by real-time PCR. Values represent the amounts of mRNA relative to those in WT littermates, which are arbitrarily defined as 1. ( E ) MMP2 activity ( Top ) was analyzed by zymography using lysates from developing lungs. The amounts of TIMP4 ( Middle ) and β-Tubulin ( Bottom ) were examined by Western blot analysis at indicated postnatal ages. ( F and G ) The expression levels of Timp4 in lungs at P6 from X/Y and X – /Y mice treated with DMSO or MEKi were examined by using real-time PCR ( F ) or Western blot analysis ( G ). ( H ) MMP2 activity levels were analyzed gelatin zymography by use of lung lysates isolated from X/Y and X – /Y mice treated with DMSO or the MEKi. In A , C , D , and F the analyses were performed with three to five independent mice per genotype. ** P

    Article Snippet: For double-staining of lungs with the antibodies for phospho-MEK1/2 and proSP-C or TIMP4 and proSP-C, antibodies were labeled using Zenon labeling kit (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Zymography, Activity Assay, Western Blot

    DA-Raf–dependent inactivation of MEK1/2 in AEC2 promotes AMYF differentiation during alveolar formation. ( A ) Distribution of phosphorylated MEK1/2 (P-MEK) was analyzed in lungs of X/Y mice at P5 by immunohistochemistry. Double-immunofluorescence staining for P-MEK (green) and proSP-C ( Upper ) or α-SMA ( Lower ) was performed. Arrows indicate representative cells with high P-MEK levels. (Scale bar, 100 μm.) ( B ) The percentages of cells containing high levels of P-MEK in lungs of X/Y (pink circles) and X – . Circles and lines indicate individual mice and median values, respectively. * P

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

    Article Title: DA-Raf–dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation

    doi: 10.1073/pnas.1321574111

    Figure Lengend Snippet: DA-Raf–dependent inactivation of MEK1/2 in AEC2 promotes AMYF differentiation during alveolar formation. ( A ) Distribution of phosphorylated MEK1/2 (P-MEK) was analyzed in lungs of X/Y mice at P5 by immunohistochemistry. Double-immunofluorescence staining for P-MEK (green) and proSP-C ( Upper ) or α-SMA ( Lower ) was performed. Arrows indicate representative cells with high P-MEK levels. (Scale bar, 100 μm.) ( B ) The percentages of cells containing high levels of P-MEK in lungs of X/Y (pink circles) and X – . Circles and lines indicate individual mice and median values, respectively. * P

    Article Snippet: For double-staining of lungs with the antibodies for phospho-MEK1/2 and proSP-C or TIMP4 and proSP-C, antibodies were labeled using Zenon labeling kit (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Mouse Assay, Immunohistochemistry, Double Immunofluorescence Staining

    DA-Raf–deficient mice have defective alveolar formation. ( A ) The protein levels of DA-Raf, Raf family members, and phosphorylated MEK1/2 in developing lungs from WT C57BL/6 mice were analyzed by Western blotting. ( B ) Sections from lungs at the indicated postnatal days of X/Y and X – /Y mice were stained with H E. Subdivision of prealveolar saccules (pas) by alveolar septa (arrowheads) at P7 results in the generation of alveoli (a) at P14 in X/Y mice. (Scale bar, 100 μm.) ( C and D ) The number of alveoli per lung ( C ) or per unit lung volume ( D ) in X/Y (pink bars) and X – at the noted postnatal stages. The values represent means ± SD of three independent mice per genotype. ** P

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

    Article Title: DA-Raf–dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation

    doi: 10.1073/pnas.1321574111

    Figure Lengend Snippet: DA-Raf–deficient mice have defective alveolar formation. ( A ) The protein levels of DA-Raf, Raf family members, and phosphorylated MEK1/2 in developing lungs from WT C57BL/6 mice were analyzed by Western blotting. ( B ) Sections from lungs at the indicated postnatal days of X/Y and X – /Y mice were stained with H E. Subdivision of prealveolar saccules (pas) by alveolar septa (arrowheads) at P7 results in the generation of alveoli (a) at P14 in X/Y mice. (Scale bar, 100 μm.) ( C and D ) The number of alveoli per lung ( C ) or per unit lung volume ( D ) in X/Y (pink bars) and X – at the noted postnatal stages. The values represent means ± SD of three independent mice per genotype. ** P

    Article Snippet: For double-staining of lungs with the antibodies for phospho-MEK1/2 and proSP-C or TIMP4 and proSP-C, antibodies were labeled using Zenon labeling kit (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Mouse Assay, Western Blot, Staining

    Analysis of the effect of chronic arsenic exposure of prostate epithelial cells on MAPK activation. ELK, E-26-like protein-1. CAsE-PE cells were first derived by exposing RWPE-1 cells to 5 μM sodium arsenite for 30 weeks. Proteins were isolated from control and arsenic-transformed CAsE-PE cells and subjected to Western blot analysis. ( A ) A- and B-Raf protein expression. ( B ) MEK1/2 and ELK-1 MAPK activation. Upper panel is a representative blot, whereas the lower panel is densitometric analysis normalized to β -actin. Densitometric data are given as fold-control and expressed as means ( n = 3); error bars represent SE. *Significantly different from control.

    Journal: Environmental Health Perspectives

    Article Title: Mechanisms of Acquired Androgen Independence during Arsenic-Induced Malignant Transformation of Human Prostate Epithelial Cells

    doi: 10.1289/ehp.9630

    Figure Lengend Snippet: Analysis of the effect of chronic arsenic exposure of prostate epithelial cells on MAPK activation. ELK, E-26-like protein-1. CAsE-PE cells were first derived by exposing RWPE-1 cells to 5 μM sodium arsenite for 30 weeks. Proteins were isolated from control and arsenic-transformed CAsE-PE cells and subjected to Western blot analysis. ( A ) A- and B-Raf protein expression. ( B ) MEK1/2 and ELK-1 MAPK activation. Upper panel is a representative blot, whereas the lower panel is densitometric analysis normalized to β -actin. Densitometric data are given as fold-control and expressed as means ( n = 3); error bars represent SE. *Significantly different from control.

    Article Snippet: Immunoblotting was performed using the human K-Ras antibody at a 1:1,000 dilution; horseradish peroxidase–conjugated anti-mouse secondary antibody at a 1:5,000 dilution; HER-2/neu antibody at 1:1,000 dilution; A-Raf antibody at 1:1,000 dilution; B-Raf antibody at 1:1,000 dilution; Phospho-MEK1/2 antibody at 1:1,000 dilution; and phospho-Elk1 antibody at 1:1,000 dilution; horseradish peroxidase–conjugated anti-rabbit secondary antibody at a 1:5,000 dilution; and SuperSignal West Pico Chemiluminescent Substrate (Pierce).

    Techniques: Activation Assay, Derivative Assay, Isolation, Transformation Assay, Western Blot, Expressing

    MAPK pathway. Abbreviations: GPCR, G protein coupled receptors; GTPase, guanosine triphosphatases. MAPK are a family of serine–threonine protein kinases widely conserved among eukaryotes and are involved in many cellular programs such as cell proliferation, cell differentiation, cell movement, and cell death. MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs (Erk) are phosphorylated and activated by MAPK-kinases (MAPKKs), (MEK1/2), which in turn are phosphorylated and activated by MAPKK-kinases (MAPKKKs), (Raf). The MAPKKKs are in turn activated by interaction with the family of small GTPases and/or other protein kinases (Ras, Rap1), connecting the MAPK module to cell surface receptors or external stimuli. An activated Erk dimer can regulate targets in the cytosol and also translocate to the nucleus where it phosphorylates a variety of transcription factors regulating gene expression.

    Journal: Environmental Health Perspectives

    Article Title: Mechanisms of Acquired Androgen Independence during Arsenic-Induced Malignant Transformation of Human Prostate Epithelial Cells

    doi: 10.1289/ehp.9630

    Figure Lengend Snippet: MAPK pathway. Abbreviations: GPCR, G protein coupled receptors; GTPase, guanosine triphosphatases. MAPK are a family of serine–threonine protein kinases widely conserved among eukaryotes and are involved in many cellular programs such as cell proliferation, cell differentiation, cell movement, and cell death. MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs (Erk) are phosphorylated and activated by MAPK-kinases (MAPKKs), (MEK1/2), which in turn are phosphorylated and activated by MAPKK-kinases (MAPKKKs), (Raf). The MAPKKKs are in turn activated by interaction with the family of small GTPases and/or other protein kinases (Ras, Rap1), connecting the MAPK module to cell surface receptors or external stimuli. An activated Erk dimer can regulate targets in the cytosol and also translocate to the nucleus where it phosphorylates a variety of transcription factors regulating gene expression.

    Article Snippet: Immunoblotting was performed using the human K-Ras antibody at a 1:1,000 dilution; horseradish peroxidase–conjugated anti-mouse secondary antibody at a 1:5,000 dilution; HER-2/neu antibody at 1:1,000 dilution; A-Raf antibody at 1:1,000 dilution; B-Raf antibody at 1:1,000 dilution; Phospho-MEK1/2 antibody at 1:1,000 dilution; and phospho-Elk1 antibody at 1:1,000 dilution; horseradish peroxidase–conjugated anti-rabbit secondary antibody at a 1:5,000 dilution; and SuperSignal West Pico Chemiluminescent Substrate (Pierce).

    Techniques: Cell Differentiation, Expressing