phospho mek1  (Millipore)


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    Name:
    MEK1
    Description:
    Mitogen activated protein kinases 1 MEK1 is encoded by the gene mapped to human chromosome 15q22 31 and the gene contains 11 exons with intervening sequences MEK1 belongs to the MEK protein family and members of this family contain kinase domain at their amino terminal and a catalytic domain at their carboxyl terminal end
    Catalog Number:
    m8697
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    Structured Review

    Millipore phospho mek1
    CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins <t>(MEK1).</t> Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p
    Mitogen activated protein kinases 1 MEK1 is encoded by the gene mapped to human chromosome 15q22 31 and the gene contains 11 exons with intervening sequences MEK1 belongs to the MEK protein family and members of this family contain kinase domain at their amino terminal and a catalytic domain at their carboxyl terminal end
    https://www.bioz.com/result/phospho mek1/product/Millipore
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 - by Bioz Stars, 2020-09
    98/100 stars

    Images

    1) Product Images from "Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers"

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

    Journal: Cancer discovery

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

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

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

    Related Articles

    Western Blot:

    Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation
    Article Snippet: .. Western blot analysis was developed using the following primary and secondary antibodies (antibodies were from Cell Signaling unless otherwise noted): anti-p53 (Ab-7; Calbiochem) (1:2,500), anti-p19ARF (Abcam) (1:400), anti-p15INK4b (Biosource) (1:400), anti-MEK1/2 (1:1,000), anti-phospho-MEK1/2 (1:1,000), anti-phospho-ERK1/2 (1:1,000), anti-Ras (EMD-Biosciences) (1:1,000), anti-caveolin-1 (610059; BD Transduction Laboratories) (1:1000), anti-KSR1 (H-70; Santa Cruz) (1:500), anti-phospho-Ser338 c-Raf (1:500), anti-c-Raf (610151; BD Transduction Laboratories) (1:500), anti-B-Raf (H-145; Santa Cruz) (1:1,000), anti-peroxisome proliferator-activated receptor γ coactivator 1α (anti-PGC-1α) (H-300; Santa Cruz) (1:1,000), anti-estrogen-related receptor α (anti-ERRα) (V-19; Santa Cruz) (1:1,000), and anti-β-actin (Sigma) (1:3,000). .. Anti-mouse, anti-rabbit, anti-sheep, and anti-goat secondary antibodies conjugated to Alexa Fluor 680 (Life Technologies) (1:3,000), IRDye 680LT (1: 20,000) (Li-Cor), or IRDye 800 (Rockland) (1:3,000) were used to probe primary antibodies.

    other:

    Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
    Article Snippet: Specific inhibitors of MEK1/2 (U0126), JNK (SP600125) and p38 (SB 203580) were purchased from Calbiochem (San Diego, CA, USA).

    Activity Assay:

    Article Title: Pan-class I  PI3-kinase inhibitor BKM120 induces MEK1/2-dependent mitotic catastrophe in non-Hodgkin lymphoma leading to apoptosis or polyploidy determined by Bax/Bak and p53
    Article Snippet: .. MEK1/2 activity was blocked with the inhibitor U0126 from Calbiochem (Merck KGaA, Darmstadt, Germany). .. Pan-caspase inhibition was performed with QV-D-OPh from Calbiochem (Merck KGaA, Darmstadt, Germany).

    Concentration Assay:

    Article Title: Induction of the Chemokines Interleukin-8 and IP-10 by Human Immunodeficiency Virus Type 1 Tat in Astrocytes
    Article Snippet: .. The p38 MAPK inhibitor SB202190 and the MEK1/2 inhibitor UO126, as well as the respective controls SB202474 and UO124 were obtained from Calbiochem (San Diego, Calif.) and dissolved in dimethyl sulfoxide to achieve a stock concentration of 20 mM. .. HIV-1 Tat72aa was produced as described earlier ( , ) and was > 98% pure.

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  • 98
    Millipore anti phospho mek1 2
    KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive <t>MEK1/2</t> and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
    Anti Phospho Mek1 2, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho mek1 2/product/Millipore
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    93
    Millipore anti mek1 phospho specific antibodies
    Identification of <t>MEK1</t> in the erythrocyte. A. Western blot of total-cell extracts from uRBCs after immunoprecipitation using either mouse anti-MEK1 agarose-conjugated or mouse IgG agarose-conjugated as a control probed with an anti-MEK1 antibody. B. Sequence Coverage of the identified MEK1 protein in erythrocyte. C. Representative CID fragmentation pattern of a unique peptide identified in MEK1.
    Anti Mek1 Phospho Specific Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mek1 phospho specific antibodies/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mek1 phospho specific antibodies - by Bioz Stars, 2020-09
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    Image Search Results


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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01633-13

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.01633-13

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

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

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

    Temporal mapping of the EGFR cascade using site-specific inhibitor molecules. The four bar graphs represent results for the following: (a) EGF stimulation, no inhibitor, and EGF stimulation in the presence of (b) neutralizing EGFR Ab, (c) GW5074—an inhibitor of Raf kinase activity, and (d) U0126—a highly selective inhibitor of MEK1/2. For each inhibitor, the arrow indicates the point of pathway inhibition. Each graph contains the time course (0–20 minutes) of expression profiles for each of the four proteins analyzed. Each bar value represents the average of three replicates.

    Journal: International Journal of Proteomics

    Article Title: Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

    doi: 10.1155/2012/473843

    Figure Lengend Snippet: Temporal mapping of the EGFR cascade using site-specific inhibitor molecules. The four bar graphs represent results for the following: (a) EGF stimulation, no inhibitor, and EGF stimulation in the presence of (b) neutralizing EGFR Ab, (c) GW5074—an inhibitor of Raf kinase activity, and (d) U0126—a highly selective inhibitor of MEK1/2. For each inhibitor, the arrow indicates the point of pathway inhibition. Each graph contains the time course (0–20 minutes) of expression profiles for each of the four proteins analyzed. Each bar value represents the average of three replicates.

    Article Snippet: As part of the initial screening process, we reviewed the certificate of analysis for each of the four antibodies: anti-phospho-EGFR (TYR1069, clone 9H2), anti-phospho-Mek1/2 (SER218/SER222, clone E237), anti-phospho-Erk 1/2 (THR202/TYR204, clone 12D4), and anti-GAPDH (clone 6C5) employed in this study (all Abs are from EMD Millipore).

    Techniques: Activity Assay, Inhibition, Expressing

    Quantitative analysis of the effects of vemurafenib, trametinib, perifosine, and regorafenib on signal transduction in CRC cell lines. The effects of (a) vemurafenib, (b) trametinib, (c) perifosine, and (d) regorafenib on fluorescence signal intensities of phosphoproteins (P-AKT, P-MEK1/2, and P-ERK1/2, resp.) and corresponding total proteins in HROC18 (black), HROC24 (grey), HROC43 (red), and HROC46 (orange) cells were quantified. Subsequently, the ratios P-MEK/MEK protein (left panels), P-ERK/ERK protein (middle panels), and P-AKT/AKT protein (right panels) were determined. A ratio of 1 corresponds to control cells cultured without SMI. Data of 5 independent experiments were used to calculate mean values ± SEM; * P

    Journal: BioMed Research International

    Article Title: Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

    doi: 10.1155/2014/568693

    Figure Lengend Snippet: Quantitative analysis of the effects of vemurafenib, trametinib, perifosine, and regorafenib on signal transduction in CRC cell lines. The effects of (a) vemurafenib, (b) trametinib, (c) perifosine, and (d) regorafenib on fluorescence signal intensities of phosphoproteins (P-AKT, P-MEK1/2, and P-ERK1/2, resp.) and corresponding total proteins in HROC18 (black), HROC24 (grey), HROC43 (red), and HROC46 (orange) cells were quantified. Subsequently, the ratios P-MEK/MEK protein (left panels), P-ERK/ERK protein (middle panels), and P-AKT/AKT protein (right panels) were determined. A ratio of 1 corresponds to control cells cultured without SMI. Data of 5 independent experiments were used to calculate mean values ± SEM; * P

    Article Snippet: The following primary antibodies (all from New England BioLabs, Frankfurt, Germany, unless specified otherwise) were employed: anti-GAPDH (#2118), anti-phospho-AKT (P-AKT; #4060), anti-phospho-MEK (P-MEK1/2; #9154), anti-phospho-ERK1/2 (P-ERK1/2) (#4370), anti-AKT protein (#4691), anti-MEK1/2 (#8727), and anti-ERK1/2 (#06-182, Millipore, Billerica, MA, United States).

    Techniques: Transduction, Fluorescence, Cell Culture

    Identification of MEK1 in the erythrocyte. A. Western blot of total-cell extracts from uRBCs after immunoprecipitation using either mouse anti-MEK1 agarose-conjugated or mouse IgG agarose-conjugated as a control probed with an anti-MEK1 antibody. B. Sequence Coverage of the identified MEK1 protein in erythrocyte. C. Representative CID fragmentation pattern of a unique peptide identified in MEK1.

    Journal: Cellular Microbiology

    Article Title: Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

    doi: 10.1111/j.1462-5822.2011.01582.x

    Figure Lengend Snippet: Identification of MEK1 in the erythrocyte. A. Western blot of total-cell extracts from uRBCs after immunoprecipitation using either mouse anti-MEK1 agarose-conjugated or mouse IgG agarose-conjugated as a control probed with an anti-MEK1 antibody. B. Sequence Coverage of the identified MEK1 protein in erythrocyte. C. Representative CID fragmentation pattern of a unique peptide identified in MEK1.

    Article Snippet: The nitrocellulose membrane was blocked for 1 h in Tris-buffered saline (pH 7.6) (TBS) containing 0.1% Tween-20 with 5% w/v non-fat dry milk and exposed overnight at 4°C to the primary antibody [1:1000 dilution in blocking buffer for anti-MEK1 (Biosource, Invitrogen) and the following anti-MEK1 phospho-specific antibodies: anti-p[S217–S221] from Calbiochem, anti-p[S217–S221] from Santa Cruz and anti-p[S297] from BioSource].

    Techniques: Western Blot, Immunoprecipitation, Sequencing

    Phosphorylation status of host cell MEK1 in P. falciparum -infected erythrocytes. A. Quantification of Western blot data obtained from Kinexus experiment (see text for details and Fig. S5 for the original Western blot). The autoradiogram was scanned to obtain a counts per minute (c.p.m.) value (scan time of 60 s). Data in Figs 4A and S5 were generated at Kinexus (Vancouver, Canada) using cell extracts provided by the authors. B. Western blot of total-cell extracts from uRBCs (lane 1) and iRBCs (lane 2). Lane 3 is a positive control extract (3T3 cells treated with PDGF) provided by the supplier of the antibodies (Biosource). The membrane was probed first with an anti-MEK1 antibody recognizing both phosphorylated and non-phosphorylated forms of the protein (left panel). The same membrane was then probed with the anti-phospho-MEK1 p[S297] (right panel). C. Effect of the PD184532 MEK inhibitor on MEK phosphorylation. Synchronous P. falciparum cultures were treated at ring stage with 20 µM PD184532 (or with the DMSO vehicle only) for 24 h prior to Western blot analysis using the antibodies indicated to the right.

    Journal: Cellular Microbiology

    Article Title: Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

    doi: 10.1111/j.1462-5822.2011.01582.x

    Figure Lengend Snippet: Phosphorylation status of host cell MEK1 in P. falciparum -infected erythrocytes. A. Quantification of Western blot data obtained from Kinexus experiment (see text for details and Fig. S5 for the original Western blot). The autoradiogram was scanned to obtain a counts per minute (c.p.m.) value (scan time of 60 s). Data in Figs 4A and S5 were generated at Kinexus (Vancouver, Canada) using cell extracts provided by the authors. B. Western blot of total-cell extracts from uRBCs (lane 1) and iRBCs (lane 2). Lane 3 is a positive control extract (3T3 cells treated with PDGF) provided by the supplier of the antibodies (Biosource). The membrane was probed first with an anti-MEK1 antibody recognizing both phosphorylated and non-phosphorylated forms of the protein (left panel). The same membrane was then probed with the anti-phospho-MEK1 p[S297] (right panel). C. Effect of the PD184532 MEK inhibitor on MEK phosphorylation. Synchronous P. falciparum cultures were treated at ring stage with 20 µM PD184532 (or with the DMSO vehicle only) for 24 h prior to Western blot analysis using the antibodies indicated to the right.

    Article Snippet: The nitrocellulose membrane was blocked for 1 h in Tris-buffered saline (pH 7.6) (TBS) containing 0.1% Tween-20 with 5% w/v non-fat dry milk and exposed overnight at 4°C to the primary antibody [1:1000 dilution in blocking buffer for anti-MEK1 (Biosource, Invitrogen) and the following anti-MEK1 phospho-specific antibodies: anti-p[S217–S221] from Calbiochem, anti-p[S217–S221] from Santa Cruz and anti-p[S297] from BioSource].

    Techniques: Infection, Western Blot, Generated, Positive Control

    Effects of PAK1 inhibitors on MEK1 phosphorylation and phosphorylation status of host erythrocyte PAK1 in P. falciparum -infected cells. A. Effect of the IPA-3 PAK1 inhibitor on MEK1 Ser-297 phosphorylation. Synchronous P. falciparum cultures were treated at ring stage with 15 µM IPA-3 (or with the DMSO vehicle only) for 27 h prior to Western blot analysis using the antibodies indicated to the right. URBC, uninfected red blood cells. B. Increased phosphorylation of PAK1 Ser-144 in iRBCs. A Western blot analysis was performed on protein extracts from in vitro cultured infected (I) or uninfected (U) RBC ghosts using anti-PAK1 antibody (Cell Signaling; top panel) or anti-phospho-PAK 1/2/3 (Ser-144) (Invitrogen; bottom panel). Lane C (positive control) contains an extract of A673 cells (Santa Cruz).

    Journal: Cellular Microbiology

    Article Title: Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

    doi: 10.1111/j.1462-5822.2011.01582.x

    Figure Lengend Snippet: Effects of PAK1 inhibitors on MEK1 phosphorylation and phosphorylation status of host erythrocyte PAK1 in P. falciparum -infected cells. A. Effect of the IPA-3 PAK1 inhibitor on MEK1 Ser-297 phosphorylation. Synchronous P. falciparum cultures were treated at ring stage with 15 µM IPA-3 (or with the DMSO vehicle only) for 27 h prior to Western blot analysis using the antibodies indicated to the right. URBC, uninfected red blood cells. B. Increased phosphorylation of PAK1 Ser-144 in iRBCs. A Western blot analysis was performed on protein extracts from in vitro cultured infected (I) or uninfected (U) RBC ghosts using anti-PAK1 antibody (Cell Signaling; top panel) or anti-phospho-PAK 1/2/3 (Ser-144) (Invitrogen; bottom panel). Lane C (positive control) contains an extract of A673 cells (Santa Cruz).

    Article Snippet: The nitrocellulose membrane was blocked for 1 h in Tris-buffered saline (pH 7.6) (TBS) containing 0.1% Tween-20 with 5% w/v non-fat dry milk and exposed overnight at 4°C to the primary antibody [1:1000 dilution in blocking buffer for anti-MEK1 (Biosource, Invitrogen) and the following anti-MEK1 phospho-specific antibodies: anti-p[S217–S221] from Calbiochem, anti-p[S217–S221] from Santa Cruz and anti-p[S297] from BioSource].

    Techniques: Infection, Indirect Immunoperoxidase Assay, Western Blot, In Vitro, Cell Culture, Positive Control