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  • 99
    Thermo Fisher b per gst fusion protein purification kit
    B Per Gst Fusion Protein Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gst fusion proteins
    <t>ERK2-mediated</t> phosphorylation of mGluR1a. a <t>GST-fusion</t> proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue ( CBB ) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min ( b–e ) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images ( b–d ) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation
    Gst Fusion Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore glutathione s transferase gst fusion proteins
    <t>ERK2-mediated</t> phosphorylation of mGluR1a. a <t>GST-fusion</t> proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue ( CBB ) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min ( b–e ) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images ( b–d ) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation
    Glutathione S Transferase Gst Fusion Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals smad3
    Reovirus activates TGF-β signaling in vivo. (A) Western blot analysis. Whole-brain lysates from mock-infected and reovirus-infected Swiss Webster pups (1,000 PFU, i.c., day 8 postinfection) were resolved on 10% polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with an antibody specific for phosphorylated <t>SMAD3</t> (pSMAD3) and with anti-β-actin. (B) Densitometry values (densitometry units/β-actin densitometry units) from six replicate individual brains per treatment group from separate litters indicate a fourfold increase in pSMAD3 expression ( P
    Smad3, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 86/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals p300
    Relationship between CBP and <t>P300</t> expression and acetylation. Scatter plots were generated for cells expressing GFP-CBP (A), HA-P300 (B), or ASF-GFP (C) to identify potential relationships between protein expression and N-Ac intensities. Linear regression analysis was performed, and R 2 values were calculated and are given for each epitope investigated. Larger R 2 values identify instances where N-Ac intensities are dependent on protein expression levels.
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    92
    Thermo Fisher gst fusion proteins
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Gst Fusion Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher glutathione s transferase gst fusion proteins
    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained <t>SDS–polyacrylamide</t> gel showing the expression and expected size of the <t>GST–polι</t> fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.
    Glutathione S Transferase Gst Fusion Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals smad1
    SCP4 dephosphorylates BMP-specific R-Smads. A , PPM1A depletion increases <t>Smad1</t> accumulation in the nucleus. C2C12 cells were stably expressed control short hairpin RNA or shPPM1A. C2C12-shPPM1A or control cells were treated with BMP2 (100 ng/ml) for 30
    Smad1, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene glutathione s transferase gst fusion proteins
    SCP4 dephosphorylates BMP-specific R-Smads. A , PPM1A depletion increases <t>Smad1</t> accumulation in the nucleus. C2C12 cells were stably expressed control short hairpin RNA or shPPM1A. C2C12-shPPM1A or control cells were treated with BMP2 (100 ng/ml) for 30
    Glutathione S Transferase Gst Fusion Proteins, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gst 96 well detection module
    SCP4 dephosphorylates BMP-specific R-Smads. A , PPM1A depletion increases <t>Smad1</t> accumulation in the nucleus. C2C12 cells were stably expressed control short hairpin RNA or shPPM1A. C2C12-shPPM1A or control cells were treated with BMP2 (100 ng/ml) for 30
    Gst 96 Well Detection Module, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare gst fusion proteins
    ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 <t>G425R</t> ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length <t>GST-SQSTM1</t> sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.
    Gst Fusion Proteins, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 13367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Syntaxin gst fusion proteins
    Effect of phosphorylation of the <t>synprint</t> N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control <t>GST,</t> GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).
    Gst Fusion Proteins, supplied by Syntaxin, used in various techniques. Bioz Stars score: 92/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc gst fusion protein
    The NH 2 -terminal SH3 domain of <t>Grb-2</t> is sufficient to bind SLP-65. Grb2-binding proteins were purified from lysates of unstimulated (lanes 1 , 3 , 5 , and 7 ) and pervanadate/H 2 O 2 -stimulated J558Lμm3 cells (lanes 2 , 4 , 6 , and 8 ) using <t>GST</t> fusion proteins containing different domains of Grb-2: NH 2 -terminal SH3 domain (lanes 1 and 2 ), NH 2 -terminal SH3 plus SH2 domain (lanes 3 and 4 ), SH2 domain (lanes 5 and 6 ), and complete Grb-2 (lanes 7 and 8 ). GST was used as a control (lanes 9 and 10 ). Proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting ( top and bottom , respectively).
    Gst Fusion Protein, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals irak3
    Rosiglitazone and not fenofibrate treatment decreases atherogenesis in obese, insulin resistant mice. ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and <t>Irak3</t> . Data are means ± SEM. Scale bar = 500 µm. * P
    Irak3, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 86/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals cyclin d1
    Rosiglitazone and not fenofibrate treatment decreases atherogenesis in obese, insulin resistant mice. ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and <t>Irak3</t> . Data are means ± SEM. Scale bar = 500 µm. * P
    Cyclin D1, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rockland Immunochemicals kip1 p27
    Upregulation of <t>Kip1/p27</t> and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)
    Kip1 P27, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti glutathione s transferase gst agarose antibody
    Upregulation of <t>Kip1/p27</t> and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)
    Anti Glutathione S Transferase Gst Agarose Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher b per gst fusion protein spin purification kit
    Upregulation of <t>Kip1/p27</t> and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)
    B Per Gst Fusion Protein Spin Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti glutathione s transferase gst antibody
    Upregulation of <t>Kip1/p27</t> and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)
    Monoclonal Anti Glutathione S Transferase Gst Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ERK2-mediated phosphorylation of mGluR1a. a GST-fusion proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue ( CBB ) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min ( b–e ) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images ( b–d ) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation

    Journal: Molecular neurobiology

    Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

    doi: 10.1007/s12035-016-0225-4

    Figure Lengend Snippet: ERK2-mediated phosphorylation of mGluR1a. a GST-fusion proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue ( CBB ) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min ( b–e ) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images ( b–d ) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation

    Article Snippet: To dephosphorylate GST-fusion proteins, we incubated GST-fusion proteins with Histagged active ERK2 (Millipore, 25 ng) in 25 µl reaction buffer containing 10 mM HEPES pH 7.4, 10 mM MgCl2 , 1 mM Na3 VO4 , 1 mM DTT, 50 µM ATP, and 2.5 µCi/tube [γ-32 P]ATP (~3000 Ci/mmol) for 30 min at 30 °C.

    Techniques: Autoradiography, Staining, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Migration

    Phosphorylation of mGluR1a-CT2 by ERK2. a Amino acid sequence analysis of mGluR1a-CT2(P1001-L1199). Eight ERK2 phosphorylation motifs (T/SP) are highlighted in red . Potential phosphorylation sites include T1064 (1), S1098 (2), T1143 (3), S1147 (4), T1151 (5), S1154 (6), S1169 (7), and T1178 (8). b A representative phosphoamino acid analysis showing GST-mGluR1a-CT2 phosphorylation at serine ( pSer ), but not threonine ( pThr ) and tyrosine ( pTyr ), residues. c, d Phosphorylation of mGluR1a-CT2 at pS/TP ( c ) and pTP ( d ) motifs by active ERK2. e Five mutants (M1–5) derived from mGluR1a-CT2(P1001-L1199). Site-directed mutants include mutations of T1064/S1098 to alanines (M1), T1064/S1098/T1143/S1147 to alanines (M2), T1064/S1098/T1143/S1147/T1151/S1154 to alanines (M3), four serines (S1098/S1147/S1154/S1169) to alanines (M4), and four threonines (T1064/T1143/T1151/T1178) to alanines (M5). e Phosphorylation of mGluR1a-CT2 mutants and WT at pS/TP by active ERK2. Note that no signal was detected in the M4 mutant. g Phosphorylation of GST-mGluR1a-CT2 and Elk-1 at PXpSP by active ERK2. Phosphorylation of CT2 and Elk-1 was detected by immunoblot ( IB ) with a phosphomotif antibody against pS/TP ( c, f ), pTP ( d ), or PXpSP ( g )

    Journal: Molecular neurobiology

    Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

    doi: 10.1007/s12035-016-0225-4

    Figure Lengend Snippet: Phosphorylation of mGluR1a-CT2 by ERK2. a Amino acid sequence analysis of mGluR1a-CT2(P1001-L1199). Eight ERK2 phosphorylation motifs (T/SP) are highlighted in red . Potential phosphorylation sites include T1064 (1), S1098 (2), T1143 (3), S1147 (4), T1151 (5), S1154 (6), S1169 (7), and T1178 (8). b A representative phosphoamino acid analysis showing GST-mGluR1a-CT2 phosphorylation at serine ( pSer ), but not threonine ( pThr ) and tyrosine ( pTyr ), residues. c, d Phosphorylation of mGluR1a-CT2 at pS/TP ( c ) and pTP ( d ) motifs by active ERK2. e Five mutants (M1–5) derived from mGluR1a-CT2(P1001-L1199). Site-directed mutants include mutations of T1064/S1098 to alanines (M1), T1064/S1098/T1143/S1147 to alanines (M2), T1064/S1098/T1143/S1147/T1151/S1154 to alanines (M3), four serines (S1098/S1147/S1154/S1169) to alanines (M4), and four threonines (T1064/T1143/T1151/T1178) to alanines (M5). e Phosphorylation of mGluR1a-CT2 mutants and WT at pS/TP by active ERK2. Note that no signal was detected in the M4 mutant. g Phosphorylation of GST-mGluR1a-CT2 and Elk-1 at PXpSP by active ERK2. Phosphorylation of CT2 and Elk-1 was detected by immunoblot ( IB ) with a phosphomotif antibody against pS/TP ( c, f ), pTP ( d ), or PXpSP ( g )

    Article Snippet: To dephosphorylate GST-fusion proteins, we incubated GST-fusion proteins with Histagged active ERK2 (Millipore, 25 ng) in 25 µl reaction buffer containing 10 mM HEPES pH 7.4, 10 mM MgCl2 , 1 mM Na3 VO4 , 1 mM DTT, 50 µM ATP, and 2.5 µCi/tube [γ-32 P]ATP (~3000 Ci/mmol) for 30 min at 30 °C.

    Techniques: Sequencing, Phosphoamino Acid Analysis, Derivative Assay, Mutagenesis

    ERK2 binding to mGluR1a. a In vitro binding assays with immobilized GST-fusion proteins containing distinct intracellular domains of mGluR1a and purified ERK2. Inactive ERK2 proteins were used in these assays. The inactive state of ERK2 was confirmed by the lack of phosphorylation signals from ERK2 proteins when tested in immunoblots ( IB ) with a phospho-specific antibody against pERK1/2. b In vitro binding assays with immobilized mGluR1a CT1 fragments and inactive or active ERK2. c GST-fusion proteins containing different fragments of mGluR1a CT1. d, e In vitro binding assays with immobilized GST-fusion mGluR1a-CT1a–c proteins and inactive ( d ) or active ( e ) ERK2. Note that CT1a but not CT1b and CT1c precipitated ERK2 and pERK2. ERK2 and pERK2 proteins bound to GST-fusion proteins were visualized with immunoblots using the specific antibodies against ERK2 and pERK1/2, respectively

    Journal: Molecular neurobiology

    Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

    doi: 10.1007/s12035-016-0225-4

    Figure Lengend Snippet: ERK2 binding to mGluR1a. a In vitro binding assays with immobilized GST-fusion proteins containing distinct intracellular domains of mGluR1a and purified ERK2. Inactive ERK2 proteins were used in these assays. The inactive state of ERK2 was confirmed by the lack of phosphorylation signals from ERK2 proteins when tested in immunoblots ( IB ) with a phospho-specific antibody against pERK1/2. b In vitro binding assays with immobilized mGluR1a CT1 fragments and inactive or active ERK2. c GST-fusion proteins containing different fragments of mGluR1a CT1. d, e In vitro binding assays with immobilized GST-fusion mGluR1a-CT1a–c proteins and inactive ( d ) or active ( e ) ERK2. Note that CT1a but not CT1b and CT1c precipitated ERK2 and pERK2. ERK2 and pERK2 proteins bound to GST-fusion proteins were visualized with immunoblots using the specific antibodies against ERK2 and pERK1/2, respectively

    Article Snippet: To dephosphorylate GST-fusion proteins, we incubated GST-fusion proteins with Histagged active ERK2 (Millipore, 25 ng) in 25 µl reaction buffer containing 10 mM HEPES pH 7.4, 10 mM MgCl2 , 1 mM Na3 VO4 , 1 mM DTT, 50 µM ATP, and 2.5 µCi/tube [γ-32 P]ATP (~3000 Ci/mmol) for 30 min at 30 °C.

    Techniques: Binding Assay, In Vitro, Purification, Western Blot

    Reovirus activates TGF-β signaling in vivo. (A) Western blot analysis. Whole-brain lysates from mock-infected and reovirus-infected Swiss Webster pups (1,000 PFU, i.c., day 8 postinfection) were resolved on 10% polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with an antibody specific for phosphorylated SMAD3 (pSMAD3) and with anti-β-actin. (B) Densitometry values (densitometry units/β-actin densitometry units) from six replicate individual brains per treatment group from separate litters indicate a fourfold increase in pSMAD3 expression ( P

    Journal: Journal of Virology

    Article Title: Reovirus Activates Transforming Growth Factor ? and Bone Morphogenetic Protein Signaling Pathways in the Central Nervous System That Contribute to Neuronal Survival following Infection ▿

    doi: 10.1128/JVI.02433-08

    Figure Lengend Snippet: Reovirus activates TGF-β signaling in vivo. (A) Western blot analysis. Whole-brain lysates from mock-infected and reovirus-infected Swiss Webster pups (1,000 PFU, i.c., day 8 postinfection) were resolved on 10% polyacrylamide gels, transferred to a nitrocellulose membrane, and probed with an antibody specific for phosphorylated SMAD3 (pSMAD3) and with anti-β-actin. (B) Densitometry values (densitometry units/β-actin densitometry units) from six replicate individual brains per treatment group from separate litters indicate a fourfold increase in pSMAD3 expression ( P

    Article Snippet: In order to identify the brain regions involved in pSMAD3 activation, we performed immunohistochemistry studies using an antibody specific to the phosphorylated form (Ser423/425) of SMAD3 (Rockland).

    Techniques: In Vivo, Western Blot, Infection, Expressing

    Relationship between CBP and P300 expression and acetylation. Scatter plots were generated for cells expressing GFP-CBP (A), HA-P300 (B), or ASF-GFP (C) to identify potential relationships between protein expression and N-Ac intensities. Linear regression analysis was performed, and R 2 values were calculated and are given for each epitope investigated. Larger R 2 values identify instances where N-Ac intensities are dependent on protein expression levels.

    Journal: Molecular and Cellular Biology

    Article Title: Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

    doi: 10.1128/MCB.23.21.7611-7627.2003

    Figure Lengend Snippet: Relationship between CBP and P300 expression and acetylation. Scatter plots were generated for cells expressing GFP-CBP (A), HA-P300 (B), or ASF-GFP (C) to identify potential relationships between protein expression and N-Ac intensities. Linear regression analysis was performed, and R 2 values were calculated and are given for each epitope investigated. Larger R 2 values identify instances where N-Ac intensities are dependent on protein expression levels.

    Article Snippet: Additionally, antibodies specifically recognizing either CBP (Ac238; Neomarkers) or P300 (Rockland) were used at 1:500 and 1:200, respectively.

    Techniques: Expressing, Generated

    Distribution of N-Ac intensities. Histograms comparing the N-Ac distributions for COS-7 cells transfected with GFP-CBP (A), HA-P300 (B), or ASF-GFP (C) with those for untransfected cells are shown. The histograms generated are representative of the results obtained for the IM and HeLa cell lines. The first y axis represents the number of untransfected cells (pink), the second y axis represents the number of transfected cells (blue), and the x axis represents the distribution range for the N-Ac intensities for the acetylated epitope indicated in the top right corner of each graph.

    Journal: Molecular and Cellular Biology

    Article Title: Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

    doi: 10.1128/MCB.23.21.7611-7627.2003

    Figure Lengend Snippet: Distribution of N-Ac intensities. Histograms comparing the N-Ac distributions for COS-7 cells transfected with GFP-CBP (A), HA-P300 (B), or ASF-GFP (C) with those for untransfected cells are shown. The histograms generated are representative of the results obtained for the IM and HeLa cell lines. The first y axis represents the number of untransfected cells (pink), the second y axis represents the number of transfected cells (blue), and the x axis represents the distribution range for the N-Ac intensities for the acetylated epitope indicated in the top right corner of each graph.

    Article Snippet: Additionally, antibodies specifically recognizing either CBP (Ac238; Neomarkers) or P300 (Rockland) were used at 1:500 and 1:200, respectively.

    Techniques: Transfection, Generated

    (A and B) Subnuclear distribution of transiently expressed GFP-CBP or HA-P300 and endogenous CBP/P300 in relation to acetylation and chromatin. Representative images are shown to highlight the ability of transiently expressed GFP-CBP (A) or HA-P300 (B) to recapitulate the expression patterns of their endogenous counterparts. Cells were paraformaldehyde fixed, costained with either anti-K5 (H4) (A) or anti-acetylated K14 (H3) (B), and counterstained with DAPI to visualize bulk chromatin. Labels on left identify cells stained for endogenous CBP or P300 (anti-CBP or anti-P300, respectively) or transiently expressing either GFP-CBP or HA-P300. Merged images depict the 3D interrelationship between either CBP, GFP-CBP, P300, or HA-P300 (green) and acetylated K5 or K14 (red) or chromatin (blue) in a single plane isolated from deconvolved z-series collected using digital imaging microscopy. (C) Example of the type of cell excluded from subsequent analysis based on morphological abnormalities (i.e., large HA-P300 aggregates concomitant with the reorganization of CBP into similar regions). HA-P300 and endogenous CBP appear green and red, respectively, in the merged image, and DAPI staining appears blue. Bars, 2 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

    doi: 10.1128/MCB.23.21.7611-7627.2003

    Figure Lengend Snippet: (A and B) Subnuclear distribution of transiently expressed GFP-CBP or HA-P300 and endogenous CBP/P300 in relation to acetylation and chromatin. Representative images are shown to highlight the ability of transiently expressed GFP-CBP (A) or HA-P300 (B) to recapitulate the expression patterns of their endogenous counterparts. Cells were paraformaldehyde fixed, costained with either anti-K5 (H4) (A) or anti-acetylated K14 (H3) (B), and counterstained with DAPI to visualize bulk chromatin. Labels on left identify cells stained for endogenous CBP or P300 (anti-CBP or anti-P300, respectively) or transiently expressing either GFP-CBP or HA-P300. Merged images depict the 3D interrelationship between either CBP, GFP-CBP, P300, or HA-P300 (green) and acetylated K5 or K14 (red) or chromatin (blue) in a single plane isolated from deconvolved z-series collected using digital imaging microscopy. (C) Example of the type of cell excluded from subsequent analysis based on morphological abnormalities (i.e., large HA-P300 aggregates concomitant with the reorganization of CBP into similar regions). HA-P300 and endogenous CBP appear green and red, respectively, in the merged image, and DAPI staining appears blue. Bars, 2 μm.

    Article Snippet: Additionally, antibodies specifically recognizing either CBP (Ac238; Neomarkers) or P300 (Rockland) were used at 1:500 and 1:200, respectively.

    Techniques: Expressing, Staining, Isolation, Imaging, Microscopy

    GFP-CBP and HA-P300 expression correlates with increased lysine 5 acetylation. Confocal micrographs depict the K5 (histone H4) acetylation status in COS-7 cells transiently expressing GFP-CBP (A), HA-P300 (B), or ASF-GFP (C). DNA is stained with DAPI (blue), acetylated K5 is identified by a secondary antibody conjugated to Cy3 that recognizes the acetylated K5 antibody (red), and GFP-CBP, HA-P300, and ASF-GFP expression is shown in green. The merge image is an overlay of the three channels. Bars, 25 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

    doi: 10.1128/MCB.23.21.7611-7627.2003

    Figure Lengend Snippet: GFP-CBP and HA-P300 expression correlates with increased lysine 5 acetylation. Confocal micrographs depict the K5 (histone H4) acetylation status in COS-7 cells transiently expressing GFP-CBP (A), HA-P300 (B), or ASF-GFP (C). DNA is stained with DAPI (blue), acetylated K5 is identified by a secondary antibody conjugated to Cy3 that recognizes the acetylated K5 antibody (red), and GFP-CBP, HA-P300, and ASF-GFP expression is shown in green. The merge image is an overlay of the three channels. Bars, 25 μm.

    Article Snippet: Additionally, antibodies specifically recognizing either CBP (Ac238; Neomarkers) or P300 (Rockland) were used at 1:500 and 1:200, respectively.

    Techniques: Expressing, Staining

    Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained SDS–polyacrylamide gel showing the expression and expected size of the GST–polι fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.

    Journal: The EMBO Journal

    Article Title: Localization of DNA polymerases ? and ? to the replication machinery is tightly co-ordinated in human cells

    doi: 10.1093/emboj/cdf618

    Figure Lengend Snippet: Fig. 4. Interaction between pol η and polι. (A and B) In vivo interaction of polη and polι using the yeast two-hybrid system. ( A ) Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7/pACT2, pGBKT7/pACT2-polι, pGBKT7-polη/pACT2 and pGBKT7-polη/pACT2-polι. A representative colony from each transformation was grown overnight at 27°C in selective medium and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 27°C for 3 days. ( B ) Determination of the minimal region of polι that interacts with polη. Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7-polη/pACT2, or (1) pGBKT7-polη/pACT2-polι, (2) pGBKT7-polη/pAR218 (N-terminal 492 residues of polι), (3) pGBKT7-polη/pAR216 (N-terminal 278 residues of polι) and (4) pGBKT7-polη/pAR220 (C-terminal 224 residues of polι). ( C ) Far-western analysis of the interaction between polι and polη. A Coomassie blue-stained SDS–polyacrylamide gel showing the expression and expected size of the GST–polι fusion proteins. A 1 µg aliquot of GST (lane 1) was used as negative control; GST–polι (lane 2); GST–Δpolι (492–715) (lane 3). The protein band indicated with an asterisk is a degradation product of the GST–Δpolι (492–715) fusion protein. Far-western blots of equivalent samples after transfer to nitrocellulose membrane and incubation of the immobilized proteins with 35 S-labelled full-length polη (lanes 4–6), polη (352–713) (lanes 5–9) and polη (595–713) (lanes 10–12). ( D ) GST pull-down assay demonstrating that polη interacts with the C-terminal region of polι. In vitro translated 35 S-labelled polη protein was incubated with glutathione–Sepharose beads and equal amounts of GST or GST–Δpolι (492–715) as indicated in Materials and methods. Bound proteins were eluted, and resolved by 4–20% SDS–PAGE. A portion of the in vitro translated 35 S-labelled polη corresponding to 10% of the labelled protein in the binding reaction was loaded as input (lane 1). The results show that polη binds GST–Δpolι (492–715) (lane 3) but not GST alone (lane 2). ( E ) Sf9 cells were infected with baculovirus supernatants containing GST–polι, His 6 -polη or both. Lysates were extracted with glutathione–Sepharose beads, and the extracted proteins analysed by SDS–PAGE and western blotting. Blots were probed with anti-polη (top) or anti-polι (middle) To check the amounts of polη in the lysates, parallel samples were extracted with nickel– agarose beads and analysed for the amount of polη by western blotting.

    Article Snippet: Purified GST fusion proteins were separated by 4–20% SDS–PAGE (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Invitrogen).

    Techniques: In Vivo, Transformation Assay, Incubation, Western Blot, Staining, Expressing, Negative Control, Pull Down Assay, In Vitro, SDS Page, Binding Assay, Infection

    SCP4 dephosphorylates BMP-specific R-Smads. A , PPM1A depletion increases Smad1 accumulation in the nucleus. C2C12 cells were stably expressed control short hairpin RNA or shPPM1A. C2C12-shPPM1A or control cells were treated with BMP2 (100 ng/ml) for 30

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation *

    doi: 10.1074/jbc.M114.568964

    Figure Lengend Snippet: SCP4 dephosphorylates BMP-specific R-Smads. A , PPM1A depletion increases Smad1 accumulation in the nucleus. C2C12 cells were stably expressed control short hairpin RNA or shPPM1A. C2C12-shPPM1A or control cells were treated with BMP2 (100 ng/ml) for 30

    Article Snippet: Rabbit anti-SCP4 polyclonal antibody was generated against the N-terminal 1–262 amino acids of SCP4 (Bethyl Laboratories), and P-Ser-206 (Smad1) antibody was raised against phospho-Ser-206 in the linker of Smad1 (Rockland Immunochemicals).

    Techniques: Stable Transfection, shRNA

    SCP4 physically interacts with Smad1. A , SCP4 interacts with Smad1 in vivo . HEK293T cells were co-transfected with Myc-Smad1, ALK3(Q233D), and a FLAG-tagged phosphatase. Smad1 was immunoprecipitated ( IP ) with anti-Myc antibody and then subjected to SDS-PAGE

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation *

    doi: 10.1074/jbc.M114.568964

    Figure Lengend Snippet: SCP4 physically interacts with Smad1. A , SCP4 interacts with Smad1 in vivo . HEK293T cells were co-transfected with Myc-Smad1, ALK3(Q233D), and a FLAG-tagged phosphatase. Smad1 was immunoprecipitated ( IP ) with anti-Myc antibody and then subjected to SDS-PAGE

    Article Snippet: Rabbit anti-SCP4 polyclonal antibody was generated against the N-terminal 1–262 amino acids of SCP4 (Bethyl Laboratories), and P-Ser-206 (Smad1) antibody was raised against phospho-Ser-206 in the linker of Smad1 (Rockland Immunochemicals).

    Techniques: In Vivo, Transfection, Immunoprecipitation, SDS Page

    SCP4 specifically dephosphorylates the S X S motif of Smad1. A , SCP4 specifically dephosphorylates Smad1 at the C-terminal S X S motif (indicated by P-Smad1), but not the Ser-206 site in the linker region. Experiment was carried out as described for

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation *

    doi: 10.1074/jbc.M114.568964

    Figure Lengend Snippet: SCP4 specifically dephosphorylates the S X S motif of Smad1. A , SCP4 specifically dephosphorylates Smad1 at the C-terminal S X S motif (indicated by P-Smad1), but not the Ser-206 site in the linker region. Experiment was carried out as described for

    Article Snippet: Rabbit anti-SCP4 polyclonal antibody was generated against the N-terminal 1–262 amino acids of SCP4 (Bethyl Laboratories), and P-Ser-206 (Smad1) antibody was raised against phospho-Ser-206 in the linker of Smad1 (Rockland Immunochemicals).

    Techniques:

    Depletion of SCP4 prolongs Smad1 phosphorylation and enhances BMP-induced Id1 expression. A , knockdown of SCP4 sustains the level of Smad1 phosphorylation. C2C12 cells were transfected with SCP4 siRNA ( siSCP4#1 ) or control siRNA ( siControl ), treated with

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation *

    doi: 10.1074/jbc.M114.568964

    Figure Lengend Snippet: Depletion of SCP4 prolongs Smad1 phosphorylation and enhances BMP-induced Id1 expression. A , knockdown of SCP4 sustains the level of Smad1 phosphorylation. C2C12 cells were transfected with SCP4 siRNA ( siSCP4#1 ) or control siRNA ( siControl ), treated with

    Article Snippet: Rabbit anti-SCP4 polyclonal antibody was generated against the N-terminal 1–262 amino acids of SCP4 (Bethyl Laboratories), and P-Ser-206 (Smad1) antibody was raised against phospho-Ser-206 in the linker of Smad1 (Rockland Immunochemicals).

    Techniques: Expressing, Transfection

    SCP4 dephosphorylates Smad1 in vitro . A , a schema of in vitro dephosphorylation assay. HEK293T cells were transfected with Myc-Smad1 (with or without HA-ALK3(Q233D)) or FLAG-SCP4 to express respective proteins. Myc-Smad1 and FLAG-SCP4 proteins were purified

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Domain (CTD) Small Phosphatase-like 2 Modulates the Canonical Bone Morphogenetic Protein (BMP) Signaling and Mesenchymal Differentiation via Smad Dephosphorylation *

    doi: 10.1074/jbc.M114.568964

    Figure Lengend Snippet: SCP4 dephosphorylates Smad1 in vitro . A , a schema of in vitro dephosphorylation assay. HEK293T cells were transfected with Myc-Smad1 (with or without HA-ALK3(Q233D)) or FLAG-SCP4 to express respective proteins. Myc-Smad1 and FLAG-SCP4 proteins were purified

    Article Snippet: Rabbit anti-SCP4 polyclonal antibody was generated against the N-terminal 1–262 amino acids of SCP4 (Bethyl Laboratories), and P-Ser-206 (Smad1) antibody was raised against phospho-Ser-206 in the linker of Smad1 (Rockland Immunochemicals).

    Techniques: In Vitro, De-Phosphorylation Assay, Transfection, Purification

    ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

    Journal: Autophagy

    Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

    doi: 10.1080/15548627.2016.1170257

    Figure Lengend Snippet: ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

    Article Snippet: Plasmids and peptides The plasmids for expression of full-length wild-type and G425R mutant SQSTM1 protein (residues 1 to 440) as GST fusion proteins (pGEX-4T-1; GE Healthcare, 28-9545-49) in E. coli have been described previously.

    Techniques: In Vitro, Sequencing, Isolation, Incubation, Western Blot

    Effect of phosphorylation of the synprint N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of the synprint N- and C-terminal halves on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with either α 1B (718–859) or α 1B (832–963) phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–859) with PKC (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , CaM KII phosphorylation of α 1B (718–859). C , PKC phosphorylation of α 1B (832–963). D , CaM KII phosphorylation of α 1B (832–963).

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Incubation, Chick Chorioallantoic Membrane Assay, SDS Page, Binding Assay

    Effect of phosphorylation of synprint polypeptides on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with α 1B (718–963) phosphorylated with PKA, PKG, PKC, or CaM KII or were treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–963) with PKA (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , PKG phosphorylation. C , PKC phosphorylation. D , CaM KII phosphorylation. E , Both PKC and CaM KII phosphorylation. Chemiluminescent signals for this and all subsequent experiments were within the linear range of the detection system.

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of synprint polypeptides on interactions with syntaxin and SNAP-25. Control GST, GST-syntaxin 1A, or GST-SNAP-25 were immobilized on glutathione-Sepharose and then incubated with α 1B (718–963) phosphorylated with PKA, PKG, PKC, or CaM KII or were treated with a control buffer without kinase. Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of α 1B (718–963) with PKA (+) or control buffer (−) followed by binding to immobilized control GST (25 kDa), GST-syntaxin 1A (60 kDa), or GST-SNAP-25 (50 kDa), and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , PKG phosphorylation. C , PKC phosphorylation. D , CaM KII phosphorylation. E , Both PKC and CaM KII phosphorylation. Chemiluminescent signals for this and all subsequent experiments were within the linear range of the detection system.

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Incubation, Chick Chorioallantoic Membrane Assay, SDS Page, Binding Assay

    Effect of phosphorylation of syntaxin and SNAP-25 on interactions with synprint polypeptides. Control GST, GST-syntaxin 1A, or GST-SNAP-25 was immobilized on glutathione-Sepharose, phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase, and then incubated with α 1B (718–963). Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of control GST (25 kDa), GST-syntaxin 1A (60 kDa), and GST-SNAP-25 (50 kDa) with PKC (+) or a control buffer (−), followed by incubation with α 1B (718–963) and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , Phosphorylation with CaM KII.

    Journal: The Journal of Neuroscience

    Article Title: Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins

    doi: 10.1523/JNEUROSCI.17-18-06929.1997

    Figure Lengend Snippet: Effect of phosphorylation of syntaxin and SNAP-25 on interactions with synprint polypeptides. Control GST, GST-syntaxin 1A, or GST-SNAP-25 was immobilized on glutathione-Sepharose, phosphorylated with either PKC or CaM KII, or treated with a control buffer without kinase, and then incubated with α 1B (718–963). Unbound reactants were removed by washing, and bound protein complexes were eluted from the matrix and separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with an anti-his-tag antibody. Membranes were then stripped and reprobed with an anti-GST antibody. A , Phosphorylation of control GST (25 kDa), GST-syntaxin 1A (60 kDa), and GST-SNAP-25 (50 kDa) with PKC (+) or a control buffer (−), followed by incubation with α 1B (718–963) and immunoblotting with an anti-his-tag antibody (α HIS ) or anti-GST antibody (α GST ). B , Phosphorylation with CaM KII.

    Article Snippet: No binding to a control GST protein (25 kDa) was observed, and anti-GST-tag immunoblotting confirmed that the phosphorylation state of the synprint polypeptide did not interfere with the immobilization of the GST fusion proteins.

    Techniques: Chick Chorioallantoic Membrane Assay, Incubation, SDS Page

    The NH 2 -terminal SH3 domain of Grb-2 is sufficient to bind SLP-65. Grb2-binding proteins were purified from lysates of unstimulated (lanes 1 , 3 , 5 , and 7 ) and pervanadate/H 2 O 2 -stimulated J558Lμm3 cells (lanes 2 , 4 , 6 , and 8 ) using GST fusion proteins containing different domains of Grb-2: NH 2 -terminal SH3 domain (lanes 1 and 2 ), NH 2 -terminal SH3 plus SH2 domain (lanes 3 and 4 ), SH2 domain (lanes 5 and 6 ), and complete Grb-2 (lanes 7 and 8 ). GST was used as a control (lanes 9 and 10 ). Proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting ( top and bottom , respectively).

    Journal: The Journal of Experimental Medicine

    Article Title: SLP-65: A New Signaling Component in B Lymphocytes which Requires Expression of the Antigen Receptor for Phosphorylation

    doi:

    Figure Lengend Snippet: The NH 2 -terminal SH3 domain of Grb-2 is sufficient to bind SLP-65. Grb2-binding proteins were purified from lysates of unstimulated (lanes 1 , 3 , 5 , and 7 ) and pervanadate/H 2 O 2 -stimulated J558Lμm3 cells (lanes 2 , 4 , 6 , and 8 ) using GST fusion proteins containing different domains of Grb-2: NH 2 -terminal SH3 domain (lanes 1 and 2 ), NH 2 -terminal SH3 plus SH2 domain (lanes 3 and 4 ), SH2 domain (lanes 5 and 6 ), and complete Grb-2 (lanes 7 and 8 ). GST was used as a control (lanes 9 and 10 ). Proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting ( top and bottom , respectively).

    Article Snippet: The GST fusion protein encompassing the complete murine Grb-2 was obtained from Upstate Biotechnology Inc.

    Techniques: Binding Assay, Purification

    Tyrosine phosphorylation of SLP-65 is dependent on BCR expression. BCR-negative J558L cells (lanes 1–3 ) and BCR-positive J558Lμm3 cells (lanes 4–6 ) were unstimulated (lanes 1 and 4 ) or stimulated with 25 μM pervanadate/H 2 O 2 either for 10 s (lanes 2 and 5 ) or 2 min (lanes 3 and 6 ). Proteins complexed with GST–Grb-2[SH3-SH2] fusion proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting ( top and bottom , respectively).

    Journal: The Journal of Experimental Medicine

    Article Title: SLP-65: A New Signaling Component in B Lymphocytes which Requires Expression of the Antigen Receptor for Phosphorylation

    doi:

    Figure Lengend Snippet: Tyrosine phosphorylation of SLP-65 is dependent on BCR expression. BCR-negative J558L cells (lanes 1–3 ) and BCR-positive J558Lμm3 cells (lanes 4–6 ) were unstimulated (lanes 1 and 4 ) or stimulated with 25 μM pervanadate/H 2 O 2 either for 10 s (lanes 2 and 5 ) or 2 min (lanes 3 and 6 ). Proteins complexed with GST–Grb-2[SH3-SH2] fusion proteins were detected by antiphosphotyrosine and anti–SLP-65 immunoblotting ( top and bottom , respectively).

    Article Snippet: The GST fusion protein encompassing the complete murine Grb-2 was obtained from Upstate Biotechnology Inc.

    Techniques: Expressing

    Rosiglitazone and not fenofibrate treatment decreases atherogenesis in obese, insulin resistant mice. ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and Irak3 . Data are means ± SEM. Scale bar = 500 µm. * P

    Journal: PLoS ONE

    Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

    doi: 10.1371/journal.pone.0062253

    Figure Lengend Snippet: Rosiglitazone and not fenofibrate treatment decreases atherogenesis in obese, insulin resistant mice. ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and Irak3 . Data are means ± SEM. Scale bar = 500 µm. * P

    Article Snippet: Irak3 in the aorta correlated positively with circulating adiponectin levels (rs = 0.41, P < 0.01) and negatively with Tnfα (rs = −0.43, P < 0.01), IL6 (rs = −0.35, P < 0.05) and Mcp1 (rs = −0.46, P < 0.001).

    Techniques: Mouse Assay, Staining, Expressing, Quantitative RT-PCR

    HFD increases atherogenesis in insulin resistant mice. Plaque volume was determined by measuring lipid (oil red O)-stained surfaces in subsequent sections; macrophages were stained with anti-Mac-3 antibody. Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Pparγ , Mcp1, Irak3 and Adipoq . Data are means ± SEM. ** P

    Journal: PLoS ONE

    Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

    doi: 10.1371/journal.pone.0062253

    Figure Lengend Snippet: HFD increases atherogenesis in insulin resistant mice. Plaque volume was determined by measuring lipid (oil red O)-stained surfaces in subsequent sections; macrophages were stained with anti-Mac-3 antibody. Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Pparγ , Mcp1, Irak3 and Adipoq . Data are means ± SEM. ** P

    Article Snippet: Irak3 in the aorta correlated positively with circulating adiponectin levels (rs = 0.41, P < 0.01) and negatively with Tnfα (rs = −0.43, P < 0.01), IL6 (rs = −0.35, P < 0.05) and Mcp1 (rs = −0.46, P < 0.001).

    Techniques: Mouse Assay, Staining, Expressing, Quantitative RT-PCR

    Adiponectin and macrophage-associated Irak3 are indispensable molecules in the anti-atherosclerotic properties of PPAR agonists. The schematic draw demonstrates the anti-atherosclerotic properties of the PPARγ agonist rosiglitazone. Treatment with rosiglitazone improves the adipocyte function characterized by a decrease in adipocyte size, a reduction in adipose tissue macrophages and an increased expression of anti-inflammatory adiponectin. The increase in blood adiponectin and de novo adiponectin production in atherosclerotic lesions is necessary for the upregulation of Irak3 in plaque macrophages, which is crucial for the indirect rosiglitazone-mediated decrease in Mcp1 secretion. Abbreviations: Mφ, macrophages; ROS, reactive oxygen species.

    Journal: PLoS ONE

    Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

    doi: 10.1371/journal.pone.0062253

    Figure Lengend Snippet: Adiponectin and macrophage-associated Irak3 are indispensable molecules in the anti-atherosclerotic properties of PPAR agonists. The schematic draw demonstrates the anti-atherosclerotic properties of the PPARγ agonist rosiglitazone. Treatment with rosiglitazone improves the adipocyte function characterized by a decrease in adipocyte size, a reduction in adipose tissue macrophages and an increased expression of anti-inflammatory adiponectin. The increase in blood adiponectin and de novo adiponectin production in atherosclerotic lesions is necessary for the upregulation of Irak3 in plaque macrophages, which is crucial for the indirect rosiglitazone-mediated decrease in Mcp1 secretion. Abbreviations: Mφ, macrophages; ROS, reactive oxygen species.

    Article Snippet: Irak3 in the aorta correlated positively with circulating adiponectin levels (rs = 0.41, P < 0.01) and negatively with Tnfα (rs = −0.43, P < 0.01), IL6 (rs = −0.35, P < 0.05) and Mcp1 (rs = −0.46, P < 0.001).

    Techniques: Expressing

    Adiponectin-induced Irak3 plays an important role in rosiglitazone-mediated decrease of Mcp1. ( A ) Soluble Mcp1 protein levels in DKO BMDM exposed to 50 µM fenofibrate, 10 µM rosiglitazone or 5 µM GW9662 for 24 hours as determined by ELISA. Data are means ± SEM; n = 16 from three different mice. $$ P

    Journal: PLoS ONE

    Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

    doi: 10.1371/journal.pone.0062253

    Figure Lengend Snippet: Adiponectin-induced Irak3 plays an important role in rosiglitazone-mediated decrease of Mcp1. ( A ) Soluble Mcp1 protein levels in DKO BMDM exposed to 50 µM fenofibrate, 10 µM rosiglitazone or 5 µM GW9662 for 24 hours as determined by ELISA. Data are means ± SEM; n = 16 from three different mice. $$ P

    Article Snippet: Irak3 in the aorta correlated positively with circulating adiponectin levels (rs = 0.41, P < 0.01) and negatively with Tnfα (rs = −0.43, P < 0.01), IL6 (rs = −0.35, P < 0.05) and Mcp1 (rs = −0.46, P < 0.001).

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay

    Upregulation of Kip1/p27 and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)

    Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

    Article Title: Upregulation of Human Glycolipid Transfer Protein (GLTP) Induces Necroptosis in Colon Carcinoma Cells

    doi: 10.1016/j.bbalip.2018.11.002

    Figure Lengend Snippet: Upregulation of Kip1/p27 and Cip1/p21 levels and modulation of CDKs by GLTP overexpression. Western blot images of HT-29 cells at 48 h post transfection with indicated vectors. ( A ) Decreased levels of Cyclin D1 and Cyclin E induced by wtGLTP overexpression are shown. By contrast, alterations are minimal with mutant GLTP (W96A) overexpression and nonexistent with GLTP depletion (siGLTP) compared to vector-control cells. ( B ) Increased levels of Kip1/p27 and Cip1/p21 and decreased levels of cyclin-dependent kinases (CDK), CDK2 and CDK4 induced by wtGLTP overexpression. Quantitative insights are provided by ratiometric comparisons of band intensities to Actin (loading ctrl.)

    Article Snippet: Kip1/p27 binds to and prevents activation of the cyclin E-CDK2 or cyclin D-CDK4 complexes, thus controlling cell cycle progression at G1 phase.

    Techniques: Over Expression, Western Blot, Transfection, Mutagenesis, Plasmid Preparation