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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 phospho erk
    Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6852 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 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
    Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 14018 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 4866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 1454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology erk
    Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of <t>PAP.</t> PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by <t>ERK</t> in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).
    Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 3862 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 phospho erk 1 2
    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Phospho Erk 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology phosphorylated erk
    Comparison of mRNA expressions and the correlations among of <t>Raf,</t> MEK, and <t>ERK.</t> A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
    Phosphorylated Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erk inhibitor pd98059
    Comparison of mRNA expressions and the correlations among of <t>Raf,</t> MEK, and <t>ERK.</t> A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
    Erk Inhibitor Pd98059, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 383 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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    Abcam extracellular signal regulated kinase
    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
    Extracellular Signal Regulated Kinase, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Papanicolaou Stain, Generated, Mutagenesis, Purification, In Vitro

    Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vivo, Transfection, Mutagenesis, Cell Culture, Activation Assay, Incubation

    Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Mass Spectrometry, Purification, In Vitro, Sequencing, Autoradiography, SDS Page, Staining

    Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vitro, Transfection, Incubation, SDS Page, Autoradiography, Expressing, Staining

    Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Activity Assay, Transfection, Papanicolaou Stain, In Vitro, Labeling, Purification, Thin Layer Chromatography

    Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Cell Culture, Immunoprecipitation

    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

    Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Papanicolaou Stain, Generated, Mutagenesis, Purification, In Vitro

    Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vivo, Transfection, Mutagenesis, Cell Culture, Activation Assay, Incubation

    Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Mass Spectrometry, Purification, In Vitro, Sequencing, Autoradiography, SDS Page, Staining

    Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vitro, Transfection, Incubation, SDS Page, Autoradiography, Expressing, Staining

    Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Activity Assay, Transfection, Papanicolaou Stain, In Vitro, Labeling, Purification, Thin Layer Chromatography

    Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Cell Culture, Immunoprecipitation

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Western Blot, Expressing

    Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques:

    Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques: Expressing

    Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques: Immunohistochemistry

    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