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  • 99
    Millipore monoclonal anti map kinase activated diphosphorylated erk 1 2 antibody
    Monoclonal Anti Map Kinase Activated Diphosphorylated Erk 1 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti erk
    Spatial relationship between VDCC and <t>ERK.</t> ( A ) Immunofluorescence images of SMGs labeled with phosphorylated ERK <t>(pERK)</t> and Ca V 1.1. ( B ) Enlarged images focusing individual buds of eSMGs. PhC: phase contrast. ( C ) Relative intensity of pERK/DAPI signals (left) and Ca V 1.1(right) of epithelial cells in the outer and inner part of eSMGs. n = 72. Data are represented as mean ±SEM. AU: arbitrary unit. ( D ) Spatial correlation between the expression levels of Ca V 1.1 and pERK signals in eSMG cultures. n = 144. ( E ) Experimental scheme for determining signaling hierarchy between ERK and VDCCs. ( F ) Intensity changes (%) in pERK and Ca V 1.1 levels in the buds of SMGs cultures upon 10 μM U0126 (left, n = 16) and 100 μM nifedipine (right, n = 10) treatment. Data are represented as mean ± SEM. ( G ) Relative intensity of G-CaMP6s and ERK (nucleus/cytoplasm) signals in SMG-C6 cells. Arrows indicate the time point of 50 mM KCl treatment. n = 25. Data are represented as mean ± SEM. ( H ) Intensity changes (%) in nuclear ERK signals by 50 mM KCl with/without 100 μM nifedipine preincubation. n = 11. Data are represented as mean ± SEM. ( I ) Representative images of SMG-C6 cells expressing RaichuEV-HRas after 50 mM KCl treatment. ( J ) Relative changes in FRET/CFP signals induced by 50 mM KCl, upon 25 μM trifluoperazine (TFP) preincubation. n = 7. Data are represented as mean ±SEM. Scale bars: 50 μm.
    Anti Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho erk
    Spatial relationship between VDCC and <t>ERK.</t> ( A ) Immunofluorescence images of SMGs labeled with phosphorylated ERK <t>(pERK)</t> and Ca V 1.1. ( B ) Enlarged images focusing individual buds of eSMGs. PhC: phase contrast. ( C ) Relative intensity of pERK/DAPI signals (left) and Ca V 1.1(right) of epithelial cells in the outer and inner part of eSMGs. n = 72. Data are represented as mean ±SEM. AU: arbitrary unit. ( D ) Spatial correlation between the expression levels of Ca V 1.1 and pERK signals in eSMG cultures. n = 144. ( E ) Experimental scheme for determining signaling hierarchy between ERK and VDCCs. ( F ) Intensity changes (%) in pERK and Ca V 1.1 levels in the buds of SMGs cultures upon 10 μM U0126 (left, n = 16) and 100 μM nifedipine (right, n = 10) treatment. Data are represented as mean ± SEM. ( G ) Relative intensity of G-CaMP6s and ERK (nucleus/cytoplasm) signals in SMG-C6 cells. Arrows indicate the time point of 50 mM KCl treatment. n = 25. Data are represented as mean ± SEM. ( H ) Intensity changes (%) in nuclear ERK signals by 50 mM KCl with/without 100 μM nifedipine preincubation. n = 11. Data are represented as mean ± SEM. ( I ) Representative images of SMG-C6 cells expressing RaichuEV-HRas after 50 mM KCl treatment. ( J ) Relative changes in FRET/CFP signals induced by 50 mM KCl, upon 25 μM trifluoperazine (TFP) preincubation. n = 7. Data are represented as mean ±SEM. Scale bars: 50 μm.
    Anti Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti erk
    Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and <t>MEK</t> and <t>ERK</t> activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.
    Anti Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho erk
    Quantification from western blots of phosphorylated to total <t>Akt</t> (A) and phosphorylated to total <t>ERK</t> (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p
    Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti phospho erk
    Fig. 6. MEK* and ΔRaf trigger <t>MAPK</t> activation when phosphatases are inhibited by okadaic acid (OA). ( A ) <t>ERK</t> immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.
    Anti Phospho Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti total erk
    Model of the role of <t>TMEFF2</t> in Akt and <t>ERK</t> activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned
    Anti Total Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti map kinase erk 1 erk 2 antibody
    Model of the role of <t>TMEFF2</t> in Akt and <t>ERK</t> activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned
    Anti Map Kinase Erk 1 Erk 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho erk thr202 tyr204
    Model of the role of <t>TMEFF2</t> in Akt and <t>ERK</t> activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned
    Anti Phospho Erk Thr202 Tyr204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 268 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibody against phospho erk
    Model of the role of <t>TMEFF2</t> in Akt and <t>ERK</t> activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned
    Antibody Against Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti erk
    Inhibition of <t>Akt</t> promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and <t>ERK</t> in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. * P
    Rabbit Anti Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti map kinase activated diphosphorylated erk 1 2 antibody mouse monoclonal
    Inhibition of <t>Akt</t> promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and <t>ERK</t> in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. * P
    Anti Map Kinase Activated Diphosphorylated Erk 1 2 Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phosphorylated erk
    Inhibition of <t>Akt</t> promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and <t>ERK</t> in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. * P
    Anti Phosphorylated Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Spatial relationship between VDCC and ERK. ( A ) Immunofluorescence images of SMGs labeled with phosphorylated ERK (pERK) and Ca V 1.1. ( B ) Enlarged images focusing individual buds of eSMGs. PhC: phase contrast. ( C ) Relative intensity of pERK/DAPI signals (left) and Ca V 1.1(right) of epithelial cells in the outer and inner part of eSMGs. n = 72. Data are represented as mean ±SEM. AU: arbitrary unit. ( D ) Spatial correlation between the expression levels of Ca V 1.1 and pERK signals in eSMG cultures. n = 144. ( E ) Experimental scheme for determining signaling hierarchy between ERK and VDCCs. ( F ) Intensity changes (%) in pERK and Ca V 1.1 levels in the buds of SMGs cultures upon 10 μM U0126 (left, n = 16) and 100 μM nifedipine (right, n = 10) treatment. Data are represented as mean ± SEM. ( G ) Relative intensity of G-CaMP6s and ERK (nucleus/cytoplasm) signals in SMG-C6 cells. Arrows indicate the time point of 50 mM KCl treatment. n = 25. Data are represented as mean ± SEM. ( H ) Intensity changes (%) in nuclear ERK signals by 50 mM KCl with/without 100 μM nifedipine preincubation. n = 11. Data are represented as mean ± SEM. ( I ) Representative images of SMG-C6 cells expressing RaichuEV-HRas after 50 mM KCl treatment. ( J ) Relative changes in FRET/CFP signals induced by 50 mM KCl, upon 25 μM trifluoperazine (TFP) preincubation. n = 7. Data are represented as mean ±SEM. Scale bars: 50 μm.

    Journal: Scientific Reports

    Article Title: Voltage-dependent Ca2+ channels promote branching morphogenesis of salivary glands by patterning differential growth

    doi: 10.1038/s41598-018-25957-w

    Figure Lengend Snippet: Spatial relationship between VDCC and ERK. ( A ) Immunofluorescence images of SMGs labeled with phosphorylated ERK (pERK) and Ca V 1.1. ( B ) Enlarged images focusing individual buds of eSMGs. PhC: phase contrast. ( C ) Relative intensity of pERK/DAPI signals (left) and Ca V 1.1(right) of epithelial cells in the outer and inner part of eSMGs. n = 72. Data are represented as mean ±SEM. AU: arbitrary unit. ( D ) Spatial correlation between the expression levels of Ca V 1.1 and pERK signals in eSMG cultures. n = 144. ( E ) Experimental scheme for determining signaling hierarchy between ERK and VDCCs. ( F ) Intensity changes (%) in pERK and Ca V 1.1 levels in the buds of SMGs cultures upon 10 μM U0126 (left, n = 16) and 100 μM nifedipine (right, n = 10) treatment. Data are represented as mean ± SEM. ( G ) Relative intensity of G-CaMP6s and ERK (nucleus/cytoplasm) signals in SMG-C6 cells. Arrows indicate the time point of 50 mM KCl treatment. n = 25. Data are represented as mean ± SEM. ( H ) Intensity changes (%) in nuclear ERK signals by 50 mM KCl with/without 100 μM nifedipine preincubation. n = 11. Data are represented as mean ± SEM. ( I ) Representative images of SMG-C6 cells expressing RaichuEV-HRas after 50 mM KCl treatment. ( J ) Relative changes in FRET/CFP signals induced by 50 mM KCl, upon 25 μM trifluoperazine (TFP) preincubation. n = 7. Data are represented as mean ±SEM. Scale bars: 50 μm.

    Article Snippet: The membranes were blocked with 10% non-fat milk and incubated with anti-ERK antibodies (1:1000; Cell Signaling Technology, 9102) and anti-pERK antibodies (1:1000; Cell signaling, 9101) at 4 °C overnight.

    Techniques: Immunofluorescence, Labeling, Expressing

    Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.

    Journal: Nature Communications

    Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

    doi: 10.1038/ncomms15262

    Figure Lengend Snippet: Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.

    Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.

    Techniques: Mouse Assay, Western Blot, Expressing, Cell Culture, Activation Assay

    RAF signalling is required for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a ) A melanoma from an untreated Braf f/f ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse was cut into small pieces and subcutaneously grafted into two groups of nude mice that were treated either with tamoxifen or vehicle for 2 weeks. The effect on tumour growth was assessed by measuring tumour volume over a 6-week period. Tumour volumes are plotted relative to the initial volume at the start of treatment. This experiment is representative of three independent experiments requiring 48 Swiss Nu/Nu females (6-week-old) for one primary tumour from a 1-year-old female on a SV129/C57Bl6 mixed genetic background. ( b ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels (pMEK and pERK, respectively) in protein lysates from culture in c on days 4 and 7 of 4OHT treatment compared to DMSO-treated culture. Total MEK, total ERK and β-actin are shown as a loading control. ( c ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ; Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour in response to 4OHT or DMSO for 9 days. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( d ) Cell cycle analysis by FACS from culture in c on day 6 of 4OHT treatment compared to DMSO-treated culture. Data are the mean value of three independent experiments. * P value

    Journal: Nature Communications

    Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

    doi: 10.1038/ncomms15262

    Figure Lengend Snippet: RAF signalling is required for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a ) A melanoma from an untreated Braf f/f ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse was cut into small pieces and subcutaneously grafted into two groups of nude mice that were treated either with tamoxifen or vehicle for 2 weeks. The effect on tumour growth was assessed by measuring tumour volume over a 6-week period. Tumour volumes are plotted relative to the initial volume at the start of treatment. This experiment is representative of three independent experiments requiring 48 Swiss Nu/Nu females (6-week-old) for one primary tumour from a 1-year-old female on a SV129/C57Bl6 mixed genetic background. ( b ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels (pMEK and pERK, respectively) in protein lysates from culture in c on days 4 and 7 of 4OHT treatment compared to DMSO-treated culture. Total MEK, total ERK and β-actin are shown as a loading control. ( c ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ; Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour in response to 4OHT or DMSO for 9 days. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( d ) Cell cycle analysis by FACS from culture in c on day 6 of 4OHT treatment compared to DMSO-treated culture. Data are the mean value of three independent experiments. * P value

    Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.

    Techniques: Mouse Assay, Western Blot, Activation Assay, Cell Culture, Cell Cycle Assay, FACS

    NRAS-mutated human melanoma cells require both BRAF and CRAF for ERK activation and proliferation. ( a ) Western blot analysis of BRAF and CRAF protein expression and pERK activation in NRAS-mutated human melanoma cell lines (WM1361, WM852 and Sbcl2) transfected with the scrambled control (−) or short interfering RNA to BRAF and/or CRAF (siBRAF1 and siCRAF1). Total ERK and β-actin are used as a loading control. ( b ) Proliferation rate in WM1361, WM852 and Sbcl2 cells transfected with scrambled control (scr), BRAF siRNA (B1), CRAF siRNA (C1) or BRAFsiRNA/CRAFsiRNA (B1C1) was measured after BrdU incorporation during 3 hours. ** P -value

    Journal: Nature Communications

    Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

    doi: 10.1038/ncomms15262

    Figure Lengend Snippet: NRAS-mutated human melanoma cells require both BRAF and CRAF for ERK activation and proliferation. ( a ) Western blot analysis of BRAF and CRAF protein expression and pERK activation in NRAS-mutated human melanoma cell lines (WM1361, WM852 and Sbcl2) transfected with the scrambled control (−) or short interfering RNA to BRAF and/or CRAF (siBRAF1 and siCRAF1). Total ERK and β-actin are used as a loading control. ( b ) Proliferation rate in WM1361, WM852 and Sbcl2 cells transfected with scrambled control (scr), BRAF siRNA (B1), CRAF siRNA (C1) or BRAFsiRNA/CRAFsiRNA (B1C1) was measured after BrdU incorporation during 3 hours. ** P -value

    Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.

    Techniques: Activation Assay, Western Blot, Expressing, Transfection, Small Interfering RNA, BrdU Incorporation Assay

    ARAF is required for the survival NRAS Q61K -induced murine melanoma cell lines in absence of BRAF and CRAF. ( a ) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( b ) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. ( c ) qRT–PCR analysis of ARAF expression in BRAF/CRAF Δ/Δ and parental control cultures. *** P value

    Journal: Nature Communications

    Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

    doi: 10.1038/ncomms15262

    Figure Lengend Snippet: ARAF is required for the survival NRAS Q61K -induced murine melanoma cell lines in absence of BRAF and CRAF. ( a ) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. ( b ) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. ( c ) qRT–PCR analysis of ARAF expression in BRAF/CRAF Δ/Δ and parental control cultures. *** P value

    Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.

    Techniques: Double Knockout, Cell Culture, Western Blot, Expressing, Activation Assay, Quantitative RT-PCR

    Vemurafenib induces ERK paradoxical activation in BRAF- and CRAF-decifient NRAS-induced melanoma by increasing ARAF kinase activity. ( a ) Western blot analysis of ERK activation (pERK) and ARAF, BRAF and CRAF protein expression in parental control and BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib (Vemu) or DMSO during 1 h. Total ERK and β-actin are used as loading controls. ( b ) ARAF in vitro kinase assays in BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib or DMSO during 1 h. ARAF was immunoprecipitated and its intrinsic kinase activity was measured on kinase-inactive MEK as substrate by western blotting using anti-pMEK antibody. Immune complexes and total cell extracts were immunoblotted with anti-ARAF, pMEK, MEK, pERK and ERK antibodies. β-actin was used as a loading control.

    Journal: Nature Communications

    Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma

    doi: 10.1038/ncomms15262

    Figure Lengend Snippet: Vemurafenib induces ERK paradoxical activation in BRAF- and CRAF-decifient NRAS-induced melanoma by increasing ARAF kinase activity. ( a ) Western blot analysis of ERK activation (pERK) and ARAF, BRAF and CRAF protein expression in parental control and BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib (Vemu) or DMSO during 1 h. Total ERK and β-actin are used as loading controls. ( b ) ARAF in vitro kinase assays in BRAF/CRAF Δ/Δ cultures after treatment with 1 μM Vemurafenib or DMSO during 1 h. ARAF was immunoprecipitated and its intrinsic kinase activity was measured on kinase-inactive MEK as substrate by western blotting using anti-pMEK antibody. Immune complexes and total cell extracts were immunoblotted with anti-ARAF, pMEK, MEK, pERK and ERK antibodies. β-actin was used as a loading control.

    Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.

    Techniques: Activation Assay, Activity Assay, Western Blot, Expressing, In Vitro, Immunoprecipitation

    Inhibition of cell proliferation and phospho-ERK expression by an ERK activation inhibitor (PD98059) in the SH-SY5Y cells. A, cells were cultured in medium without fetal bovine serum for 24 h and then with 10% fetal bovine serum in the presence of various concentrations of PD98059 for 2 days. Cell numbers were then measured and normalized by those at the beginning of the fetal bovine serum stimulation. B–D , cells treated as described for panel A were harvested for Western blotting of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3 (pSMAD2/3), total SMAD2/3 (tSMAD2/3), and phospho-SMAD1/5/8 (pSMAD1/5/8). The ERK and SMAD Western blot results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). The results from cells incubated with PD98059 were then normalized by those from control cells. Results are mean ± S.D. ( n = 3–4). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor ?2 Transcription in Human Neuroblastoma Cells *

    doi: 10.1074/jbc.M111.338590

    Figure Lengend Snippet: Inhibition of cell proliferation and phospho-ERK expression by an ERK activation inhibitor (PD98059) in the SH-SY5Y cells. A, cells were cultured in medium without fetal bovine serum for 24 h and then with 10% fetal bovine serum in the presence of various concentrations of PD98059 for 2 days. Cell numbers were then measured and normalized by those at the beginning of the fetal bovine serum stimulation. B–D , cells treated as described for panel A were harvested for Western blotting of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3 (pSMAD2/3), total SMAD2/3 (tSMAD2/3), and phospho-SMAD1/5/8 (pSMAD1/5/8). The ERK and SMAD Western blot results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). The results from cells incubated with PD98059 were then normalized by those from control cells. Results are mean ± S.D. ( n = 3–4). *, p

    Article Snippet: After being blocked with the Protein-Free T20 Blocking Buffer (Thermo Scientific), membranes were incubated with each of the following primary antibodies: E-16 anti-RFX1 antibody (Santa Cruz Biotechnology), anti-phospho-ERK antibody (Cell Signaling Technology, Danvers, MA), anti-ERK antibody (Santa Cruz Biotechnology), anti-phospho-SMAD2 (Cell Signaling Technology), anti-phospho-SMAD3 (Cell Signaling Technology), anti-SMAD2/3 antibody (Cell Signaling Technology), anti-phospho-SMAD1/5/8 antibody (Cell Signaling Technology), and anti-GAPDH antibody (Sigma).

    Techniques: Inhibition, Expressing, Activation Assay, Cell Culture, Western Blot, Incubation

    Reduction of TGFβ2 and phospho-ERK expression by RFX1 in the SH-SY5Y cells. A, real-time PCR results of TGF β1, TGF β2, TGF β receptor 1 ( TGF β R1 ), and TGF β R2 . These results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and actin. The results from the RFX1 overexpressing cells were then normalized by those from control cells. B , TGFβ2 concentrations in the culture medium included with the cells for 4 days. C, real-time PCR results of fibroblast growth factor 1 ( FGF1 ) and FGF1 protein concentrations in the culture medium included with the cells for 4 days. D, a representative Western blot image of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3, and total SMAD2/3 expression in the RFX1 overexpressing cells and control cells. E , quantification of protein expression of pERK, tERK, phospho-SMAD2/3 (pSMAD2/3), and total SMAD2/3 (tSMAD2/3). The ERK and SMAD Western blot results were normalized by those of GAPDH. The results from the RFX1 overexpressing cells were then normalized by those from control cells. F , protein expression of phospho-SMAD1/5/8 (pSMAD1/5/8). The normalization process was identical to that described for panel E . Results are mean ± S.D. ( n = 3–6). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor ?2 Transcription in Human Neuroblastoma Cells *

    doi: 10.1074/jbc.M111.338590

    Figure Lengend Snippet: Reduction of TGFβ2 and phospho-ERK expression by RFX1 in the SH-SY5Y cells. A, real-time PCR results of TGF β1, TGF β2, TGF β receptor 1 ( TGF β R1 ), and TGF β R2 . These results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and actin. The results from the RFX1 overexpressing cells were then normalized by those from control cells. B , TGFβ2 concentrations in the culture medium included with the cells for 4 days. C, real-time PCR results of fibroblast growth factor 1 ( FGF1 ) and FGF1 protein concentrations in the culture medium included with the cells for 4 days. D, a representative Western blot image of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3, and total SMAD2/3 expression in the RFX1 overexpressing cells and control cells. E , quantification of protein expression of pERK, tERK, phospho-SMAD2/3 (pSMAD2/3), and total SMAD2/3 (tSMAD2/3). The ERK and SMAD Western blot results were normalized by those of GAPDH. The results from the RFX1 overexpressing cells were then normalized by those from control cells. F , protein expression of phospho-SMAD1/5/8 (pSMAD1/5/8). The normalization process was identical to that described for panel E . Results are mean ± S.D. ( n = 3–6). *, p

    Article Snippet: After being blocked with the Protein-Free T20 Blocking Buffer (Thermo Scientific), membranes were incubated with each of the following primary antibodies: E-16 anti-RFX1 antibody (Santa Cruz Biotechnology), anti-phospho-ERK antibody (Cell Signaling Technology, Danvers, MA), anti-ERK antibody (Santa Cruz Biotechnology), anti-phospho-SMAD2 (Cell Signaling Technology), anti-phospho-SMAD3 (Cell Signaling Technology), anti-SMAD2/3 antibody (Cell Signaling Technology), anti-phospho-SMAD1/5/8 antibody (Cell Signaling Technology), and anti-GAPDH antibody (Sigma).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Inhibition of cell proliferation and phospho-ERK expression by an anti-TGFβ2 antibody in the SH-SY5Y cells. A, a dot blot produced by loading 0.1 or 1 ng of TGFβ2 or FGF1 on the membrane and visualizing the proteins by an anti-TGFβ2 antibody. B, cells were cultured in medium without fetal bovine serum for 24 h and then with 10% fetal bovine serum in the presence of various concentrations of an anti-TGFβ2 antibody for 2 days. Cell numbers then were measured and normalized by those at the beginning of fetal bovine serum stimulation. C–E, cells treated as described for panel B were harvested for Western blotting of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3 (pSMAD2/3), total SMAD2/3 (tSMAD2/3), and phospho-SMAD1/5/8 (pSMAD1/5/8). The ERK and SMAD Western blot results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). The results from cells incubated with the anti-TGFβ2 antibody were then normalized by those from control cells. Results are mean ± S.D. ( n = 3–4). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Regulatory Factor X1-induced Down-regulation of Transforming Growth Factor ?2 Transcription in Human Neuroblastoma Cells *

    doi: 10.1074/jbc.M111.338590

    Figure Lengend Snippet: Inhibition of cell proliferation and phospho-ERK expression by an anti-TGFβ2 antibody in the SH-SY5Y cells. A, a dot blot produced by loading 0.1 or 1 ng of TGFβ2 or FGF1 on the membrane and visualizing the proteins by an anti-TGFβ2 antibody. B, cells were cultured in medium without fetal bovine serum for 24 h and then with 10% fetal bovine serum in the presence of various concentrations of an anti-TGFβ2 antibody for 2 days. Cell numbers then were measured and normalized by those at the beginning of fetal bovine serum stimulation. C–E, cells treated as described for panel B were harvested for Western blotting of phospho-ERK (pERK), total ERK (tERK), phospho-SMAD2/3 (pSMAD2/3), total SMAD2/3 (tSMAD2/3), and phospho-SMAD1/5/8 (pSMAD1/5/8). The ERK and SMAD Western blot results were normalized by those of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). The results from cells incubated with the anti-TGFβ2 antibody were then normalized by those from control cells. Results are mean ± S.D. ( n = 3–4). *, p

    Article Snippet: After being blocked with the Protein-Free T20 Blocking Buffer (Thermo Scientific), membranes were incubated with each of the following primary antibodies: E-16 anti-RFX1 antibody (Santa Cruz Biotechnology), anti-phospho-ERK antibody (Cell Signaling Technology, Danvers, MA), anti-ERK antibody (Santa Cruz Biotechnology), anti-phospho-SMAD2 (Cell Signaling Technology), anti-phospho-SMAD3 (Cell Signaling Technology), anti-SMAD2/3 antibody (Cell Signaling Technology), anti-phospho-SMAD1/5/8 antibody (Cell Signaling Technology), and anti-GAPDH antibody (Sigma).

    Techniques: Inhibition, Expressing, Dot Blot, Produced, Cell Culture, Western Blot, Incubation

    Quantification from western blots of phosphorylated to total Akt (A) and phosphorylated to total ERK (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p

    Journal: Journal of cardiovascular pharmacology

    Article Title: Cardiac Outcomes after Perinatal Sertraline Exposure in Mice

    doi: 10.1097/FJC.0000000000000501

    Figure Lengend Snippet: Quantification from western blots of phosphorylated to total Akt (A) and phosphorylated to total ERK (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p

    Article Snippet: Primary antibodies for β-actin (1:10000), total (1:5000) and phosphorylated Akt (1:1000) and total (1:2000) and phosphorylated ERK (1:2000)(Cell Signaling Technology, Danvers, MA) were assessed.

    Techniques: Western Blot, Isolation, Mouse Assay

    Western blot analysis after β-escin treatment. HUVECs were pre-treated with the indicated concentration of β-escin and then stimulated with 30 ng/mL of bFGF for 30 min before collection. Phosphorylated Akt, ERK 1/2 or p38 were detected by specific antibodies. The pictures shown are representative of three independent experiments. Western blots were quantified by densitometry and the ratio of phosphorylated Akt, phosphorylated ERK 1/2 or phosphorylated p38 to their total counterpart was expressed as mean ± SD of three experiments (***  p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: β-Escin Effectively Modulates HUVECs Proliferation and Tube Formation

    doi: 10.3390/molecules23010197

    Figure Lengend Snippet: Western blot analysis after β-escin treatment. HUVECs were pre-treated with the indicated concentration of β-escin and then stimulated with 30 ng/mL of bFGF for 30 min before collection. Phosphorylated Akt, ERK 1/2 or p38 were detected by specific antibodies. The pictures shown are representative of three independent experiments. Western blots were quantified by densitometry and the ratio of phosphorylated Akt, phosphorylated ERK 1/2 or phosphorylated p38 to their total counterpart was expressed as mean ± SD of three experiments (*** p

    Article Snippet: The following primary antibodies were used: anti-phospho-ERK 1/2 (Cell Signaling Technology, Beverly, MA, USA, 1:2000), anti-phospho-Akt (Cell Signaling Technology, 1:2000) and anti-phospho-p38 (Cell Signaling Technology, 1:1000).

    Techniques: Western Blot, Concentration Assay

    Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Activation Assay, Injection, Cell Culture, Kinase Assay

    Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture, Incubation

    Fig. 8. The co-injection of MEK* and Xp42 mapk D324N in mos –/– oocytes rescues the M II arrest. ( A ) Mos –/– oocytes were either not injected (control), injected with RNAs encoding Xp42 mapk D324N alone (xMAPK*), co-injected with wild-type MycERK2 and constitutively active MEK* (MAPK + MEK*), or co-injected with RNAs encoding Xp42 mapk D324N and constitutively active MEK* (xMAPK* + MEK*) in dbcAMP-containing medium. After 5 h incubation in dbcAMP to allow overexpression of the exogenous proteins, the oocytes were transferred to M2 medium for overnight culture. The oocytes were then scored for the presence of polar bodies and normal bipolar spindles (see B): 0 or 1 polar body with abnormal spindles (arrested in M I or M II, abnormal spindles); one polar body and a normal spindle (M II); or two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. ( B ) Immunofluorescence staining of microtubules and chromatin in mos –/– oocytes microinjected with MEK* and Xp42 mapk D324N (MEK* + xMAPK*) or Xp42 mapk D324N alone (xMAPK*). Oocytes were microinjected in dbcAMP and kept in the drug for 5 h. The oocytes were transferred to M2 medium for overnight culture and fixed 16 h after GVBD. Microtubules appear in green and chromosomes in red. Top: normal spindles in oocytes injected with MEK* and Xp42 mapk D324N that extruded one polar body (M II). Bottom: abnormal spindles in oocytes injected with Xp42 mapk D324N that did not extrude the first polar body (M I, left) or extruded only one polar body (M II, right). ( C ) M II-arrested mos –/– oocytes co-injected with MEK* and Xp42 mapk D324N show Xp42 mapk D324N phosphorylation whereas the activated ones do not. Oocytes treated as in (A) were collected separately, i.e. M II-arrested (M II, lane 1) and activated (PB2, lane 2). Groups of 15 oocytes were immunoblotted with the anti-ERK serum. These results correspond to two independent experiments. The MycERK2 is overexpressed after co-injection with MEK* into mos –/– oocytes. Fifty mos –/– oocytes either not injected (NI, lane 3) or co-injected with MEK* and MycERK2 (MEK* + MycERK2, lane 4) were scored after second polar body extrusion, then collected and subjected to immunoblotting using the anti-Myc antibody.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 8. The co-injection of MEK* and Xp42 mapk D324N in mos –/– oocytes rescues the M II arrest. ( A ) Mos –/– oocytes were either not injected (control), injected with RNAs encoding Xp42 mapk D324N alone (xMAPK*), co-injected with wild-type MycERK2 and constitutively active MEK* (MAPK + MEK*), or co-injected with RNAs encoding Xp42 mapk D324N and constitutively active MEK* (xMAPK* + MEK*) in dbcAMP-containing medium. After 5 h incubation in dbcAMP to allow overexpression of the exogenous proteins, the oocytes were transferred to M2 medium for overnight culture. The oocytes were then scored for the presence of polar bodies and normal bipolar spindles (see B): 0 or 1 polar body with abnormal spindles (arrested in M I or M II, abnormal spindles); one polar body and a normal spindle (M II); or two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. ( B ) Immunofluorescence staining of microtubules and chromatin in mos –/– oocytes microinjected with MEK* and Xp42 mapk D324N (MEK* + xMAPK*) or Xp42 mapk D324N alone (xMAPK*). Oocytes were microinjected in dbcAMP and kept in the drug for 5 h. The oocytes were transferred to M2 medium for overnight culture and fixed 16 h after GVBD. Microtubules appear in green and chromosomes in red. Top: normal spindles in oocytes injected with MEK* and Xp42 mapk D324N that extruded one polar body (M II). Bottom: abnormal spindles in oocytes injected with Xp42 mapk D324N that did not extrude the first polar body (M I, left) or extruded only one polar body (M II, right). ( C ) M II-arrested mos –/– oocytes co-injected with MEK* and Xp42 mapk D324N show Xp42 mapk D324N phosphorylation whereas the activated ones do not. Oocytes treated as in (A) were collected separately, i.e. M II-arrested (M II, lane 1) and activated (PB2, lane 2). Groups of 15 oocytes were immunoblotted with the anti-ERK serum. These results correspond to two independent experiments. The MycERK2 is overexpressed after co-injection with MEK* into mos –/– oocytes. Fifty mos –/– oocytes either not injected (NI, lane 3) or co-injected with MEK* and MycERK2 (MEK* + MycERK2, lane 4) were scored after second polar body extrusion, then collected and subjected to immunoblotting using the anti-Myc antibody.

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Injection, Incubation, Over Expression, Immunofluorescence, Staining

    Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture

    Model of the role of TMEFF2 in Akt and ERK activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned

    Journal: International Journal of Biochemistry and Molecular Biology

    Article Title: TMEFF2 modulates the AKT and ERK signaling pathways

    doi:

    Figure Lengend Snippet: Model of the role of TMEFF2 in Akt and ERK activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned

    Article Snippet: Other antibodies were as follow: anti-TMEFF2 (Abcam, Cambridge, MA), anti-phospho-ERK1/2, anti-total-ERK, anti-phospho-Akt (Ser473), anti-total-Akt, anti-phospho-Smad2 (Cell Signaling Technology, Danvers, MA).

    Techniques: Activation Assay

    The TMEFF2 ectodomain promotes AKT phosphorylation and that inversely correlates with its effect on ERK phosphorylation. RWPE-1 cells transferred to basal KSF medium for 4 hours before the medium was replaced with ectodomain containing conditioned medium

    Journal: International Journal of Biochemistry and Molecular Biology

    Article Title: TMEFF2 modulates the AKT and ERK signaling pathways

    doi:

    Figure Lengend Snippet: The TMEFF2 ectodomain promotes AKT phosphorylation and that inversely correlates with its effect on ERK phosphorylation. RWPE-1 cells transferred to basal KSF medium for 4 hours before the medium was replaced with ectodomain containing conditioned medium

    Article Snippet: Other antibodies were as follow: anti-TMEFF2 (Abcam, Cambridge, MA), anti-phospho-ERK1/2, anti-total-ERK, anti-phospho-Akt (Ser473), anti-total-Akt, anti-phospho-Smad2 (Cell Signaling Technology, Danvers, MA).

    Techniques:

    Conditioned medium from HEK293T cells expressing the ectodomain construct contains secreted TMEFF2 ectodomain and inhibits ERK phosphorylation. A: Exponentially growing HEK293T cells transfected with the TMEFF2-ectodomain construct or the empty vector

    Journal: International Journal of Biochemistry and Molecular Biology

    Article Title: TMEFF2 modulates the AKT and ERK signaling pathways

    doi:

    Figure Lengend Snippet: Conditioned medium from HEK293T cells expressing the ectodomain construct contains secreted TMEFF2 ectodomain and inhibits ERK phosphorylation. A: Exponentially growing HEK293T cells transfected with the TMEFF2-ectodomain construct or the empty vector

    Article Snippet: Other antibodies were as follow: anti-TMEFF2 (Abcam, Cambridge, MA), anti-phospho-ERK1/2, anti-total-ERK, anti-phospho-Akt (Ser473), anti-total-Akt, anti-phospho-Smad2 (Cell Signaling Technology, Danvers, MA).

    Techniques: Expressing, Construct, Transfection, Plasmid Preparation

    PDGF induces sustained phosphorylation of ERK in cells expressing TMEFF2. A: RWPE-1 cells were transferred to basal KSF medium for 30 minutes and then treated with various concentrations of PDGF-AA or 50 ng/ml of EGF. Whole cell lysates were then subjected

    Journal: International Journal of Biochemistry and Molecular Biology

    Article Title: TMEFF2 modulates the AKT and ERK signaling pathways

    doi:

    Figure Lengend Snippet: PDGF induces sustained phosphorylation of ERK in cells expressing TMEFF2. A: RWPE-1 cells were transferred to basal KSF medium for 30 minutes and then treated with various concentrations of PDGF-AA or 50 ng/ml of EGF. Whole cell lysates were then subjected

    Article Snippet: Other antibodies were as follow: anti-TMEFF2 (Abcam, Cambridge, MA), anti-phospho-ERK1/2, anti-total-ERK, anti-phospho-Akt (Ser473), anti-total-Akt, anti-phospho-Smad2 (Cell Signaling Technology, Danvers, MA).

    Techniques: Expressing

    Inhibition of Akt promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. * P

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

    doi: 10.1371/journal.pone.0113555

    Figure Lengend Snippet: Inhibition of Akt promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control) or 0.1, 1 or 10 µM Akt inhibitor VIII over 48 hours. Images representative of at least 12 explants for each condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. * P

    Article Snippet: Proteins were subsequently transferred to a nitrocellulose membrane and probed with either rabbit anti-phospho-Akt (Ser473) (Cat No: 4060S; Cell Signaling), rabbit anti phospho-S6 ribosomal protein (Ser235/236)(Cat No: 2211S; Cell Signaling), rabbit anti-Akt (Cat No: 9272; Cell Signaling) or rabbit anti-ERK (Cat No: SC-93; Santa Cruz, Middlesex, UK).

    Techniques: Inhibition, Light Microscopy, Cell Culture, Isolation, Western Blot

    Inhibition of PI3K promotes epithelial branching in murine lung explants. A. Light microscope images of E12.5 murine lung explants cultured with either 0.1% DMSO (Control) or 0.1, 1 or 10 µM ZSTK474 over 48 hours. Images representative of at least 12 explants per condition are shown. Scale bar = 0.5 mm B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 20 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. D. Confocal image of E-Cadherin expression in lung explants cultured with or without 10 µM ZSTK474 for 48 hours. Scale bar = 200 µm. *** P

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

    doi: 10.1371/journal.pone.0113555

    Figure Lengend Snippet: Inhibition of PI3K promotes epithelial branching in murine lung explants. A. Light microscope images of E12.5 murine lung explants cultured with either 0.1% DMSO (Control) or 0.1, 1 or 10 µM ZSTK474 over 48 hours. Images representative of at least 12 explants per condition are shown. Scale bar = 0.5 mm B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 20 C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. D. Confocal image of E-Cadherin expression in lung explants cultured with or without 10 µM ZSTK474 for 48 hours. Scale bar = 200 µm. *** P

    Article Snippet: Proteins were subsequently transferred to a nitrocellulose membrane and probed with either rabbit anti-phospho-Akt (Ser473) (Cat No: 4060S; Cell Signaling), rabbit anti phospho-S6 ribosomal protein (Ser235/236)(Cat No: 2211S; Cell Signaling), rabbit anti-Akt (Cat No: 9272; Cell Signaling) or rabbit anti-ERK (Cat No: SC-93; Santa Cruz, Middlesex, UK).

    Techniques: Inhibition, Light Microscopy, Cell Culture, Isolation, Western Blot, Expressing

    Inhibition of mTORC2 but not mTORC1 promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured in either 0.1% DMSO (Control), 1 µM rapamycin or 0.1 µM AZD8055. Images representative of at least 12 explants per condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. C. Western blot analysis for levels of phosphorylated S6 ribosomal protein (p-S6), phosphorylated Akt (p-Akt), total Akt and ERK in explants cultured for 48 hours. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. *** P

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

    doi: 10.1371/journal.pone.0113555

    Figure Lengend Snippet: Inhibition of mTORC2 but not mTORC1 promotes epithelial branching. A. Light microscope images of E12.5 murine lung explants cultured in either 0.1% DMSO (Control), 1 µM rapamycin or 0.1 µM AZD8055. Images representative of at least 12 explants per condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. C. Western blot analysis for levels of phosphorylated S6 ribosomal protein (p-S6), phosphorylated Akt (p-Akt), total Akt and ERK in explants cultured for 48 hours. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. *** P

    Article Snippet: Proteins were subsequently transferred to a nitrocellulose membrane and probed with either rabbit anti-phospho-Akt (Ser473) (Cat No: 4060S; Cell Signaling), rabbit anti phospho-S6 ribosomal protein (Ser235/236)(Cat No: 2211S; Cell Signaling), rabbit anti-Akt (Cat No: 9272; Cell Signaling) or rabbit anti-ERK (Cat No: SC-93; Santa Cruz, Middlesex, UK).

    Techniques: Inhibition, Light Microscopy, Cell Culture, Isolation, Western Blot

    The alpha isoform of PI3K regulates lung branching morphogenesis. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control), 10 µM BYL719 or 10 µM GSK2636771. Images representative of at least 12 explants per condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. *** P

    Journal: PLoS ONE

    Article Title: Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

    doi: 10.1371/journal.pone.0113555

    Figure Lengend Snippet: The alpha isoform of PI3K regulates lung branching morphogenesis. A. Light microscope images of E12.5 murine lung explants cultured with 0.1% DMSO (Control), 10 µM BYL719 or 10 µM GSK2636771. Images representative of at least 12 explants per condition are shown. B. Percentage increase in epithelial branching over 24 and 48 hours relative to the number of branches at initial isolation. Bars show mean ± s.e.m from n = 12. C. Western blot analysis for levels of phosphorylated Akt (p-Akt), total Akt and ERK in explants following 48 hours of culture. Total Akt and phosphorylated Akt bands are visualised from different gels containing an equal amount of the same protein lysate. Protein bands representative of 3 separate experiments. Scale bar = 0.5 mm. *** P

    Article Snippet: Proteins were subsequently transferred to a nitrocellulose membrane and probed with either rabbit anti-phospho-Akt (Ser473) (Cat No: 4060S; Cell Signaling), rabbit anti phospho-S6 ribosomal protein (Ser235/236)(Cat No: 2211S; Cell Signaling), rabbit anti-Akt (Cat No: 9272; Cell Signaling) or rabbit anti-ERK (Cat No: SC-93; Santa Cruz, Middlesex, UK).

    Techniques: Light Microscopy, Cell Culture, Isolation, Western Blot