γ-32 p atp Search Results


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  • 99
    Thermo Fisher γ 32 p atp
    γ 32 P Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore γ 32 p adenosine triphosphate
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    Valiant γ 32 p atp
    SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P <t>ATP.</t> The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.
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    DuPont de Nemours γ 32 p atp
    Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 <t>P]ATP</t> and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.
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    HARTMANN ANALYTIC γ 32 p atp
    Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 <t>P]-ATP</t> labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.
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    NEN Life Science γ 32 p atp
    15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras  cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras  cells were treated with 15d-PGJ 2  in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras  cells. The stained cells were analyzed under a confocal microscope.
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    PerkinElmer γ 32 p atp
    15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras  cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras  cells were treated with 15d-PGJ 2  in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras  cells. The stained cells were analyzed under a confocal microscope.
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    ICN Biomedicals γ 32 p atp
    Tyrosine phosphorylation of PKR by Jaks. ( A ) 2fTGH and U4A cells were stimulated with IFN-α2b for the indicated time periods. Protein extracts were immunoprecipitated (IP) for PKR followed by immunoblotting with phosphotyrosine 4G10 antibody (panel a), PKR monoclonal antibody (panel b) and eIF2α antibody (panel c). The levels of PKR (panel d) and eIF2α (panel e) in WCE were detected by immunoblotting. ( B ) U4A cells were transfected with the catalytically inactive Flag–PKRK296R complementary DNA in the absence or presence of either wild-type (WT) or a kinase-dead (KD) K296R mutant of Jak1 cDNA. Protein extracts were immunoprecipitated for Flag followed by immunoblotting with phosphotyrosine 4G10 (panel b) or PKR antibody (panel c). Jak1 levels (panel a) were detected by immunoblotting of WCE. ( C ) Recombinant murine Jak2 and GST–PKRK296R were subjected to in vitro phosphorylation with [γ- 32 <t>P]ATP</t> and autoradiography (panels a and b). The phosphorylated proteins were detected after an equal time of exposure. Total Jak2 (panel c) and GST–PKRK296R (panel d) levels were detected by Coomassie blue staining. IFN, interferon; PKR, dsRNA-dependent protein kinase; PKRK296R, PKRLys296Arg; WCE, whole-cell extracts.
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    Boehringer Mannheim γ 32 p atp
    Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 <t>P]ATP.</t> Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).
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    Bio-Rad γ 32 p atp
    Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 <t>P]ATP.</t> Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).
    γ 32 P Atp, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer atp γ 32p lead
    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P <t>ATP</t> (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).
    Atp γ 32p Lead, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Pharmaceuticals γ 32 p atp
    TG induces JNK activation in a time- and dose-dependent manner. Jurkat T lymphocytes were treated with TG for various times. The cell lysates were prepared and immunoprecipitated with 10 μg of polyclonal anti-JNK1 antibody followed by 20 μl of Sepharose A-conjugated protein A. The kinase reaction was performed by the procedures described in Materials and Methods. (A) Time course of the kinase reaction. The top figure represents the autoradiogram of [γ- 32 <t>P]ATP</t> incorporation into exogenous GST–c-Jun-(1–135). The amount of cell lysate used was 200 μg of protein in each lane. The bottom figure is a plot of JNK activity against time. The values in this figure are means and standard errors of three determinations. (B) Dose response of JNK activation. Anti-JNK1 immunocomplexes were obtained with lysates of cells treated with various doses of TG for 3 h. The JNK assay was performed as described in Materials and Methods. The top figure is a representative autoradiogram of three independent experiments. The bottom figure shows quantified data from three determinations (means and standard errors).
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    Muromachi Kikai γ 32 p atp
    Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] <t>ATP</t> as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.
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    PerkinElmer atp γ 32p easytide
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
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    Promega γ 32 p atp
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
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    PerkinElmer atp γ 32p easytide lead
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
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    GeneWorks γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
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    Image Search Results


    SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.

    Journal: Nucleic Acids Research

    Article Title: Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A

    doi: 10.1093/nar/gkr195

    Figure Lengend Snippet: SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.

    Article Snippet: The ECL kit was from Thermo Scientific (Rockford, IL, USA), and [γ-32 P] ATP and [32 P] orthophosphate were from MP Biomedicals (Irvine, CA, USA).

    Techniques: Western Blot, Immunoprecipitation, ALP Assay, Incubation, Labeling, Nucleic Acid Electrophoresis, Transfection, Reverse Transcription Polymerase Chain Reaction

    Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Generated, Mutagenesis, Construct, Incubation, Affinity Chromatography, SDS Page, Autoradiography, Molecular Weight

    Fig. 4. Phosphorylation inhibits the in vitro interaction of 1a and 2a proteins. ( A ) Schematic representation of the GSTK-1a and GSTK-2a fusion proteins. The pentapeptide substrate RRASV, which is a substrate for the catalytic subunit of cAMP-dependent protein kinase (PKA), was introduced into the 3′ end of the GST gene. ( B ) Phosphorylation analysis of the GSTK fusion proteins. GSTK-2a was phosphorylated in vitro using [γ- 32 P]ATP and either t2akI (lane 1) or PKA (lane 2), and GSTK-1a was phosphorylated in vitro using [γ- 32 P]ATP and PKA (lane 3). ( C ) Co-immunoprecipitation analysis of phosphorylated GSTK-2a and GSTK-1a fusions proteins. GSTK-2a, phosphorylated in vitro by either t2akI (lane 1) or PKA (lane 2), was mixed with unlabeled GST–1a, and incubated with anti-1a serum. GSTK-1a phosphorylated in vitro by PKA (lane 3), was mixed with carrier, unlabeled GST–1a and immunoprecipitated by incubation with anti-1a serum. The reaction mixtures in (B) and the immunoprecipitates formed in (C) were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 4. Phosphorylation inhibits the in vitro interaction of 1a and 2a proteins. ( A ) Schematic representation of the GSTK-1a and GSTK-2a fusion proteins. The pentapeptide substrate RRASV, which is a substrate for the catalytic subunit of cAMP-dependent protein kinase (PKA), was introduced into the 3′ end of the GST gene. ( B ) Phosphorylation analysis of the GSTK fusion proteins. GSTK-2a was phosphorylated in vitro using [γ- 32 P]ATP and either t2akI (lane 1) or PKA (lane 2), and GSTK-1a was phosphorylated in vitro using [γ- 32 P]ATP and PKA (lane 3). ( C ) Co-immunoprecipitation analysis of phosphorylated GSTK-2a and GSTK-1a fusions proteins. GSTK-2a, phosphorylated in vitro by either t2akI (lane 1) or PKA (lane 2), was mixed with unlabeled GST–1a, and incubated with anti-1a serum. GSTK-1a phosphorylated in vitro by PKA (lane 3), was mixed with carrier, unlabeled GST–1a and immunoprecipitated by incubation with anti-1a serum. The reaction mixtures in (B) and the immunoprecipitates formed in (C) were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Immunoprecipitation, Incubation, SDS Page, Autoradiography, Molecular Weight

    Fig. 3. Localization of the C-terminal in vitro phosphorylation domain of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. Regions phosphorylated are shown in black, while regions not phosphorylated are shown in gray. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose. Samples: GST–2a(334–515) (lane 1); GST–2a (408–685) (lane 2); GST–2a(408–605) (lane 3); and GST–2a(517–685) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 3. Localization of the C-terminal in vitro phosphorylation domain of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. Regions phosphorylated are shown in black, while regions not phosphorylated are shown in gray. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose. Samples: GST–2a(334–515) (lane 1); GST–2a (408–685) (lane 2); GST–2a(408–605) (lane 3); and GST–2a(517–685) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Generated, Mutagenesis, Construct, Incubation, Affinity Chromatography, SDS Page, Autoradiography, Molecular Weight

    Fig. 1. Phosphorylation of CMV 2a protein in vivo and in vitro . ( A ) In vivo phosphorylation. Tobacco protoplasts were either not infected (lanes 1, 3, 5, 7 and 9) or infected with total CMV-As RNAs (lanes 2, 4, 6, 8 and 10) by electroporation. The protoplasts were incubated for various times in the presence of [ 32 P]orthophosphate. At the time intervals indicated in hours post-inoculation (h.p.i.), protoplasts were harvested, lysed and proteins were immunoprecipitated using anti-2a antibody. The immunoprecipitated proteins were analyzed by SDS–PAGE and autoradiography. The position of the 2a protein is indicated by an arrow. ( B ) In vitro phosphorylation. Proteins were solubilized from tobacco membranes and fractionated by chromatography on Q-Sepharose. The bound fraction eluting with 1 M NaCl was used as a source of protein kinases in an in vitro phosphorylation reaction with [γ- 32 P]ATP, using GST–2a (lane 1), free GST (lane 2) or no added protein (lane 3) as a substrate. The reaction mixtures were analyzed by SDS–PAGE and autoradiography. ( C ) In vitro phosphorylation. Three tobacco kinases—t2akI (p60), t2akII (p55) and t2akIII (p35)—obtained by hydrophobic interaction chromatography on phenyl–Sepharose followed by anion-exchange chromatography on Q-Sepharose (lane 1), were further fractionated by affinity chromatography on heparin– Sepharose (lanes 2–5). After the flow-through (lane 2), the three protein kinases were each eluted stepwise from the heparin–Sepharose matrix: 0.1–0.6 M NaCl eluate (lane 3); 0.6 M NaCl eluate (lane 4); and 1.5 M eluate (lane 5). Equal volumes of column eluate were used for each assay, and equal volumes of reaction mixture were analyzed by SDS–PAGE and autoradiography. The position of the phosphorylated GST–2a protein is indicated by an arrow in (B) and (C). The positions of molecular weight standards are indicated on the left.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 1. Phosphorylation of CMV 2a protein in vivo and in vitro . ( A ) In vivo phosphorylation. Tobacco protoplasts were either not infected (lanes 1, 3, 5, 7 and 9) or infected with total CMV-As RNAs (lanes 2, 4, 6, 8 and 10) by electroporation. The protoplasts were incubated for various times in the presence of [ 32 P]orthophosphate. At the time intervals indicated in hours post-inoculation (h.p.i.), protoplasts were harvested, lysed and proteins were immunoprecipitated using anti-2a antibody. The immunoprecipitated proteins were analyzed by SDS–PAGE and autoradiography. The position of the 2a protein is indicated by an arrow. ( B ) In vitro phosphorylation. Proteins were solubilized from tobacco membranes and fractionated by chromatography on Q-Sepharose. The bound fraction eluting with 1 M NaCl was used as a source of protein kinases in an in vitro phosphorylation reaction with [γ- 32 P]ATP, using GST–2a (lane 1), free GST (lane 2) or no added protein (lane 3) as a substrate. The reaction mixtures were analyzed by SDS–PAGE and autoradiography. ( C ) In vitro phosphorylation. Three tobacco kinases—t2akI (p60), t2akII (p55) and t2akIII (p35)—obtained by hydrophobic interaction chromatography on phenyl–Sepharose followed by anion-exchange chromatography on Q-Sepharose (lane 1), were further fractionated by affinity chromatography on heparin– Sepharose (lanes 2–5). After the flow-through (lane 2), the three protein kinases were each eluted stepwise from the heparin–Sepharose matrix: 0.1–0.6 M NaCl eluate (lane 3); 0.6 M NaCl eluate (lane 4); and 1.5 M eluate (lane 5). Equal volumes of column eluate were used for each assay, and equal volumes of reaction mixture were analyzed by SDS–PAGE and autoradiography. The position of the phosphorylated GST–2a protein is indicated by an arrow in (B) and (C). The positions of molecular weight standards are indicated on the left.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vivo, In Vitro, Infection, Electroporation, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography, Flow Cytometry, Molecular Weight

    Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 P]-ATP labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 P]-ATP labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: In Vitro, Construct, Labeling, Negative Control

    Type I-A Cascade cleaves ssDNA unspecifically and is inhibited by crRNA. ( A ) Cascade (0, 0.05, 0.125, 0.25, 0.5 µM) was incubated with a 5′-[γ- 32 P]-ATP labeled ssDNA fragment (int_5.2 CCT for) in the presence of Mg 2+ and Mn 2+ ions and the nucleolytic cleavage reaction was resolved on 15% denaturing gels. After 1–10-min incubation at a fixed Cascade concentration of 0.1 µM, > 70% of the substrate is cleaved. In the presence of 0.5 µM unlabeled crRNA, the reaction is inhibited. ( B ) This observation was tested in the presence of 0, 0.05, 0.5 and 2.5 µM crRNA, and the amount of remaining substrate estimated via line profile plots (Image J) was plotted for three different reaction times (1, 2, 10 min) and reactions performed in triplicate. ( C ) Cas3′′ subunit with HD domain mutations (H19A, H55A, D56A) was assembled into Cascade and then tested in ssDNA cleavage assays.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Type I-A Cascade cleaves ssDNA unspecifically and is inhibited by crRNA. ( A ) Cascade (0, 0.05, 0.125, 0.25, 0.5 µM) was incubated with a 5′-[γ- 32 P]-ATP labeled ssDNA fragment (int_5.2 CCT for) in the presence of Mg 2+ and Mn 2+ ions and the nucleolytic cleavage reaction was resolved on 15% denaturing gels. After 1–10-min incubation at a fixed Cascade concentration of 0.1 µM, > 70% of the substrate is cleaved. In the presence of 0.5 µM unlabeled crRNA, the reaction is inhibited. ( B ) This observation was tested in the presence of 0, 0.05, 0.5 and 2.5 µM crRNA, and the amount of remaining substrate estimated via line profile plots (Image J) was plotted for three different reaction times (1, 2, 10 min) and reactions performed in triplicate. ( C ) Cas3′′ subunit with HD domain mutations (H19A, H55A, D56A) was assembled into Cascade and then tested in ssDNA cleavage assays.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: Incubation, Labeling, Concentration Assay

    Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: RNA Binding Assay, Recombinant, Incubation, Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Binding Assay, Labeling, Purification, Construct, Clear Native PAGE

    Interference activity of in vitro assembled type I-A Cascade. ( A ) The assembled Cascade complex is loaded with crRNA 5.2 for 20 min at 70°C, and the interference reaction is started with the addition of ATP, Mg 2+ , Mn 2+ and the dsDNA substrate (in_5.2 CCT), which is either 5′-[γ- 32 P]-ATP labeled on the non-target (forward) or the crRNA target strand (reverse). Cleavage reactions were stopped at three different time points (1, 5, 10 min at 70°C). The reaction products of the cleaved dsDNA were separated on 20% denaturing gels. The non-matching crRNA 5.13 and the Cas3′′ D56A mutant are used as controls. ( B ) In parallel, the crRNA 5.13 is loaded into Cascade, and cleavage of the matching dsDNA substrate (in_5.13 CCT) is visualized. ( C and D ) The cleavage products are analyzed on 10% Urea-PAGE for each strand (in_5.2 CCT for/rev and in_5.13 CCT for/rev) with two different markers ( Supplementary Figure S9 ). The cleavage sites are marked within the proposed R-loop structure that is formed during the interference reaction [(C) dsDNA 5.2, (D) dsDNA 5.13].

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Interference activity of in vitro assembled type I-A Cascade. ( A ) The assembled Cascade complex is loaded with crRNA 5.2 for 20 min at 70°C, and the interference reaction is started with the addition of ATP, Mg 2+ , Mn 2+ and the dsDNA substrate (in_5.2 CCT), which is either 5′-[γ- 32 P]-ATP labeled on the non-target (forward) or the crRNA target strand (reverse). Cleavage reactions were stopped at three different time points (1, 5, 10 min at 70°C). The reaction products of the cleaved dsDNA were separated on 20% denaturing gels. The non-matching crRNA 5.13 and the Cas3′′ D56A mutant are used as controls. ( B ) In parallel, the crRNA 5.13 is loaded into Cascade, and cleavage of the matching dsDNA substrate (in_5.13 CCT) is visualized. ( C and D ) The cleavage products are analyzed on 10% Urea-PAGE for each strand (in_5.2 CCT for/rev and in_5.13 CCT for/rev) with two different markers ( Supplementary Figure S9 ). The cleavage sites are marked within the proposed R-loop structure that is formed during the interference reaction [(C) dsDNA 5.2, (D) dsDNA 5.13].

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: Activity Assay, In Vitro, Labeling, Mutagenesis, Polyacrylamide Gel Electrophoresis

    15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras  cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras  cells were treated with 15d-PGJ 2  in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras  cells. The stained cells were analyzed under a confocal microscope.

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: 15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras cells were treated with 15d-PGJ 2 in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras cells. The stained cells were analyzed under a confocal microscope.

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Binding Assay, Activity Assay, Isolation, Incubation, Labeling, Sequencing, Staining, Microscopy

    Downregulation of IKKβ is associated with induction of apoptosis in MCF10A- ras  cells treated with 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ). (A, B) MCF10A- ras  cells were cotreated with N -acetyl-L-cysteine (NAC) (5 mM) or a thiol reducing agent, dithiothreitol (DTT) (500 μM), in the presence of 15d-PGJ 2  (10 μM) for 24 hours. The expression level of IKKβ was determined by Western blot analysis. The proteins were immunoprecipitated by anti-IKKβ and subsequently incubated with glutathione S-transferase-IκB-a and [γ- 32 P]ATP for the kinase assay. Murine immunoglobulin G heavy chain band was used to ensure the equal lane loading. (C) MCF10A- ras  cells were transfected with IKKβ and its mutant construct in which Cys179 is replaced by alanine. The catalytic activity of IKKβ and proteolytic cleavage of caspase-3 were determined by the kinase assay and Western blot analysis, respectively. (D) MCF10A- ras  cells were treated with biotinylated 15d-PGJ 2  (10 μM) for 12 hours. The biotinylated 15d-PGJ 2 -IKKβ complex was detected by the immunoprecipitation with IKKβ followed by Western blot analysis against horseradish peroxidase (HRP)-streptavidin as described in Materials and Methods.

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: Downregulation of IKKβ is associated with induction of apoptosis in MCF10A- ras cells treated with 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ). (A, B) MCF10A- ras cells were cotreated with N -acetyl-L-cysteine (NAC) (5 mM) or a thiol reducing agent, dithiothreitol (DTT) (500 μM), in the presence of 15d-PGJ 2 (10 μM) for 24 hours. The expression level of IKKβ was determined by Western blot analysis. The proteins were immunoprecipitated by anti-IKKβ and subsequently incubated with glutathione S-transferase-IκB-a and [γ- 32 P]ATP for the kinase assay. Murine immunoglobulin G heavy chain band was used to ensure the equal lane loading. (C) MCF10A- ras cells were transfected with IKKβ and its mutant construct in which Cys179 is replaced by alanine. The catalytic activity of IKKβ and proteolytic cleavage of caspase-3 were determined by the kinase assay and Western blot analysis, respectively. (D) MCF10A- ras cells were treated with biotinylated 15d-PGJ 2 (10 μM) for 12 hours. The biotinylated 15d-PGJ 2 -IKKβ complex was detected by the immunoprecipitation with IKKβ followed by Western blot analysis against horseradish peroxidase (HRP)-streptavidin as described in Materials and Methods.

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Transfection, Mutagenesis, Construct, Activity Assay

    The cyclopentenone ring on 15-deoxy-Δ 12,14 -prostaglandin J 2  (15d-PGJ 2 ) is critical for 15d-PGJ 2 -induced apoptosis in MCF10A- ras cells. (A) The chemical structures of 15d-PGJ 2  and its non-electrophilic 9,10-dihydro-15d-PGJ 2 . An asterisk indicates an electrophilic carbon. (B, C) MCF10A- ras  cells were treated with 15d-PGJ 2  (10 μM) or 9,10-dihydro-15d-PGJ 2  (10 μM) for 24 hours. Reactive oxygen species was measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining. (C) Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB consensus sequence. The DNA binding activity were measured by electrophoretic mobility shifty assay. (D) MCF10A- ras  cells were treated with 15d-PGJ 2  (10 μM) or 9,10-dihydro-15d-PGJ 2  (10 μM) for 24 hours, and cytotoxicity was measured by the MTT assay. Bars represent mean ± SE of three independent assays. A significant difference in the relative viability between treated cells and the solvent control is indicated with P

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: The cyclopentenone ring on 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) is critical for 15d-PGJ 2 -induced apoptosis in MCF10A- ras cells. (A) The chemical structures of 15d-PGJ 2 and its non-electrophilic 9,10-dihydro-15d-PGJ 2 . An asterisk indicates an electrophilic carbon. (B, C) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) or 9,10-dihydro-15d-PGJ 2 (10 μM) for 24 hours. Reactive oxygen species was measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining. (C) Nuclear extracts from MCF10A- ras cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB consensus sequence. The DNA binding activity were measured by electrophoretic mobility shifty assay. (D) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) or 9,10-dihydro-15d-PGJ 2 (10 μM) for 24 hours, and cytotoxicity was measured by the MTT assay. Bars represent mean ± SE of three independent assays. A significant difference in the relative viability between treated cells and the solvent control is indicated with P

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Staining, Incubation, Labeling, Sequencing, Binding Assay, Activity Assay, MTT Assay

    Tyrosine phosphorylation of PKR by Jaks. ( A ) 2fTGH and U4A cells were stimulated with IFN-α2b for the indicated time periods. Protein extracts were immunoprecipitated (IP) for PKR followed by immunoblotting with phosphotyrosine 4G10 antibody (panel a), PKR monoclonal antibody (panel b) and eIF2α antibody (panel c). The levels of PKR (panel d) and eIF2α (panel e) in WCE were detected by immunoblotting. ( B ) U4A cells were transfected with the catalytically inactive Flag–PKRK296R complementary DNA in the absence or presence of either wild-type (WT) or a kinase-dead (KD) K296R mutant of Jak1 cDNA. Protein extracts were immunoprecipitated for Flag followed by immunoblotting with phosphotyrosine 4G10 (panel b) or PKR antibody (panel c). Jak1 levels (panel a) were detected by immunoblotting of WCE. ( C ) Recombinant murine Jak2 and GST–PKRK296R were subjected to in vitro phosphorylation with [γ- 32 P]ATP and autoradiography (panels a and b). The phosphorylated proteins were detected after an equal time of exposure. Total Jak2 (panel c) and GST–PKRK296R (panel d) levels were detected by Coomassie blue staining. IFN, interferon; PKR, dsRNA-dependent protein kinase; PKRK296R, PKRLys296Arg; WCE, whole-cell extracts.

    Journal: EMBO Reports

    Article Title: Interferons induce tyrosine phosphorylation of the eIF2? kinase PKR through activation of Jak1 and Tyk2

    doi: 10.1038/sj.embor.7400891

    Figure Lengend Snippet: Tyrosine phosphorylation of PKR by Jaks. ( A ) 2fTGH and U4A cells were stimulated with IFN-α2b for the indicated time periods. Protein extracts were immunoprecipitated (IP) for PKR followed by immunoblotting with phosphotyrosine 4G10 antibody (panel a), PKR monoclonal antibody (panel b) and eIF2α antibody (panel c). The levels of PKR (panel d) and eIF2α (panel e) in WCE were detected by immunoblotting. ( B ) U4A cells were transfected with the catalytically inactive Flag–PKRK296R complementary DNA in the absence or presence of either wild-type (WT) or a kinase-dead (KD) K296R mutant of Jak1 cDNA. Protein extracts were immunoprecipitated for Flag followed by immunoblotting with phosphotyrosine 4G10 (panel b) or PKR antibody (panel c). Jak1 levels (panel a) were detected by immunoblotting of WCE. ( C ) Recombinant murine Jak2 and GST–PKRK296R were subjected to in vitro phosphorylation with [γ- 32 P]ATP and autoradiography (panels a and b). The phosphorylated proteins were detected after an equal time of exposure. Total Jak2 (panel c) and GST–PKRK296R (panel d) levels were detected by Coomassie blue staining. IFN, interferon; PKR, dsRNA-dependent protein kinase; PKRK296R, PKRLys296Arg; WCE, whole-cell extracts.

    Article Snippet: The agarose-bound Jak2 was resuspended in 15 μl of kinase buffer containing 10 μCi [γ-32 P]ATP (ICN Biomedicals Inc., Irvine, CA, USA) and 10 ng of purified GST-PKRK296R, and the kinase reaction was carried out at 30°C for 30 min. Proteins were subjected to SDS–polyacrylamide gel electrophoresis and autoradiography.

    Techniques: Immunoprecipitation, Transfection, Mutagenesis, Recombinant, In Vitro, Autoradiography, Staining

    Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).

    Journal: Molecular and Cellular Biology

    Article Title: Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

    doi:

    Figure Lengend Snippet: Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).

    Article Snippet: Kinase reactions were started by adding 30 μl of a mixture containing 1 mM dithiothreitol, 5 μM ATP, 20 μCi of [γ-32 P]ATP, and 3 μg of histone 2B (H2B; Boehringer Mannheim) in kinase reaction buffer.

    Techniques: Activation Assay, Transient Transfection Assay, Plasmid Preparation, Activity Assay, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Expressing, Transfection, Lysis

    Differential effects of R-Ras effector loop mutants on the activation of Akt. (A) A kinase transient-transfection assay was performed by transfecting COS-7 cells with 5 μg of the indicated plasmids together with 1 μg of an HA-tagged Akt plasmid. Akt activity was measured with H2B as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of H2B was determined by densitometric analysis and is expressed as fold activation relative to the control (Ctr). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-Akt. The expression levels of the AU5-tagged R-Ras mutants in the transfected cells were examined with an anti-AU5 monoclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type; WB, Western blot. (B) The effect of wortmannin on R-Ras-induced Akt activity was assessed by similar kinase assays except that wortmannin at the indicated concentrations was added to the culture medium 30 min prior to the kinase assay. DMSO, dimethyl sulfoxide.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

    doi:

    Figure Lengend Snippet: Differential effects of R-Ras effector loop mutants on the activation of Akt. (A) A kinase transient-transfection assay was performed by transfecting COS-7 cells with 5 μg of the indicated plasmids together with 1 μg of an HA-tagged Akt plasmid. Akt activity was measured with H2B as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of H2B was determined by densitometric analysis and is expressed as fold activation relative to the control (Ctr). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-Akt. The expression levels of the AU5-tagged R-Ras mutants in the transfected cells were examined with an anti-AU5 monoclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type; WB, Western blot. (B) The effect of wortmannin on R-Ras-induced Akt activity was assessed by similar kinase assays except that wortmannin at the indicated concentrations was added to the culture medium 30 min prior to the kinase assay. DMSO, dimethyl sulfoxide.

    Article Snippet: Kinase reactions were started by adding 30 μl of a mixture containing 1 mM dithiothreitol, 5 μM ATP, 20 μCi of [γ-32 P]ATP, and 3 μg of histone 2B (H2B; Boehringer Mannheim) in kinase reaction buffer.

    Techniques: Activation Assay, Transient Transfection Assay, Plasmid Preparation, Activity Assay, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Expressing, Transfection, Western Blot, Kinase Assay

    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P ATP (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).

    Journal: Nucleic Acids Research

    Article Title: Cyclin-dependent kinase 1 activity coordinates the chromatin associated state of Oct4 during cell cycle in embryonic stem cells

    doi: 10.1093/nar/gky371

    Figure Lengend Snippet: Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P ATP (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).

    Article Snippet: For radioactive in vitro kinase assay, (His)6 -PP1 and GST-Oct4 were incubated with Cdk1/CyclinB1 in kinase buffer (60 mM HEPES–NaOH pH 7.5, 3 mM MgCl2 , 3 mM MnCl2 , 3 mM Na-orthovanadate, 1.2 mM DTT, 0.25 mM ATP) with 0.1 mM γ-32 P-ATP (NEG002A250UC, purchased from PerkinElmer, Waltham, MA, USA) for 30 min at 30°C.

    Techniques: Staining, Activity Assay, Immunostaining, Fluorescence, Immunoprecipitation, Expressing, FACS, In Vitro, Kinase Assay, Recombinant, Mutagenesis, IP-Kinase Assay, Western Blot

    TG induces JNK activation in a time- and dose-dependent manner. Jurkat T lymphocytes were treated with TG for various times. The cell lysates were prepared and immunoprecipitated with 10 μg of polyclonal anti-JNK1 antibody followed by 20 μl of Sepharose A-conjugated protein A. The kinase reaction was performed by the procedures described in Materials and Methods. (A) Time course of the kinase reaction. The top figure represents the autoradiogram of [γ- 32 P]ATP incorporation into exogenous GST–c-Jun-(1–135). The amount of cell lysate used was 200 μg of protein in each lane. The bottom figure is a plot of JNK activity against time. The values in this figure are means and standard errors of three determinations. (B) Dose response of JNK activation. Anti-JNK1 immunocomplexes were obtained with lysates of cells treated with various doses of TG for 3 h. The JNK assay was performed as described in Materials and Methods. The top figure is a representative autoradiogram of three independent experiments. The bottom figure shows quantified data from three determinations (means and standard errors).

    Journal: Molecular and Cellular Biology

    Article Title: Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

    doi:

    Figure Lengend Snippet: TG induces JNK activation in a time- and dose-dependent manner. Jurkat T lymphocytes were treated with TG for various times. The cell lysates were prepared and immunoprecipitated with 10 μg of polyclonal anti-JNK1 antibody followed by 20 μl of Sepharose A-conjugated protein A. The kinase reaction was performed by the procedures described in Materials and Methods. (A) Time course of the kinase reaction. The top figure represents the autoradiogram of [γ- 32 P]ATP incorporation into exogenous GST–c-Jun-(1–135). The amount of cell lysate used was 200 μg of protein in each lane. The bottom figure is a plot of JNK activity against time. The values in this figure are means and standard errors of three determinations. (B) Dose response of JNK activation. Anti-JNK1 immunocomplexes were obtained with lysates of cells treated with various doses of TG for 3 h. The JNK assay was performed as described in Materials and Methods. The top figure is a representative autoradiogram of three independent experiments. The bottom figure shows quantified data from three determinations (means and standard errors).

    Article Snippet: [γ-32 P]ATP (specific activity, 3,000 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc. (Irvine, Calif.).

    Techniques: Activation Assay, Immunoprecipitation, Activity Assay

    Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.

    Journal: Marine Drugs

    Article Title: Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia

    doi: 10.3390/md10040744

    Figure Lengend Snippet: Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.

    Article Snippet: The reaction was carried out at 37 °C for 10 min in 10 μL of the reaction mixture containing 25 mM MOPS-NaOH (pH 7.0), 1 mM DTT, 5 mM MgCl2 , 5 mM CaCl2 , 1 mM [γ-32 P] ATP (Muromachi, Tokyo, Japan), 300 nM NS3, and 0.1 μg poly (U) per microliter and an indicated concentration of SG1-23-1, and then was terminated by the addition of 15 microliters of 10 mM EDTA.

    Techniques: RNA Binding Assay, Incubation, Thin Layer Chromatography, Mobility Shift, Activity Assay, Concentration Assay

    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 P-ATP. a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.

    Journal: Nature

    Article Title: AUTS2 confers gene activation to Polycomb group proteins in the CNS

    doi: 10.1038/nature13921

    Figure Lengend Snippet: H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 P-ATP. a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.

    Article Snippet: CK2 (NEB, P6010) was then added at 300 nM along with 2 µCi γ-32 P-ATP (PerkinElmer, BLU502H500UC), followed by incubation at 37°C for 30 min.

    Techniques: Kinase Assay, Staining, Incubation, SDS Page, Labeling, Purification

    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 P]ATP, while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.

    Journal: The EMBO Journal

    Article Title: Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation

    doi: 10.1093/emboj/cdg540

    Figure Lengend Snippet: Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 P]ATP, while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.

    Article Snippet: Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol) and [γ-32 P]ATP (Geneworks) as substrates, as described previously ( ).

    Techniques: In Vitro, Activation Assay, Recombinant, Mutagenesis, Staining, Quantitation Assay, Activity Assay