γ-32 p atp Perkinelmer Search Results


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  • 79
    PerkinElmer γ 32 p atp perkinelmer waltham ma
    γ 32 P Atp Perkinelmer Waltham Ma, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer atp γ 32 p
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Atp γ 32 P, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    PerkinElmer radioisotope
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Radioisotope, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 89/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer adenosine triphosphate
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Adenosine Triphosphate, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer radioactive γ 32 p atp
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Radioactive γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer radiolabelled γ 32 p atp
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Radiolabelled γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer nuclease assay γ 32 p atp
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Nuclease Assay γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer phosphorylation mix
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Phosphorylation Mix, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer kinase reaction buffer
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Kinase Reaction Buffer, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    PerkinElmer ci mmol γ 32 p atp
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Ci Mmol γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 83/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer kbq γ
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Kbq γ, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    PerkinElmer radionucleotide γ 32 p atp
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Radionucleotide γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer cpm nmol
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
    Cpm Nmol, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 83/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer mbq γ
    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] <t>ATP</t> for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
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    PerkinElmer m atp
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    M Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer radioactive atp
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    Radioactive Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer 6000 ci mmol γ 32 p atp
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    6000 Ci Mmol γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer c p m nmol γ 32 p atp
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    C P M Nmol γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer easytides
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    Easytides, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
    Atp Solution, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer adenosine 5 γ 32 p triphosphate
    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ <t>γ</t> - 32 <t>P]ATP.</t> Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment
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    Image Search Results


    Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.

    Journal: Open biology

    Article Title: Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

    doi: 10.1098/rsob.110012

    Figure Lengend Snippet: Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.

    Article Snippet: [γ-32 P] ATP was from PerkinElmer (Waltham, MA).

    Techniques: Mutagenesis, Activity Assay, Incubation, Staining

    Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.

    Journal: Open biology

    Article Title: Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

    doi: 10.1098/rsob.110012

    Figure Lengend Snippet: Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.

    Article Snippet: [γ-32 P] ATP was from PerkinElmer (Waltham, MA).

    Techniques: Activity Assay, Incubation, Staining, SDS Page, Autoradiography, In Vivo, Mutagenesis

    Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.

    Journal: Experimental & Molecular Medicine

    Article Title: Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

    doi: 10.1038/emm.2016.13

    Figure Lengend Snippet: Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.

    Article Snippet: Materials [γ-32 P] ATP was purchased from Perkin-Elmer (Waltham, MA, USA), SuperSignal West Pico Chemiluminescent substrate kit from Thermo Fisher Scientific Inc. (Waltham, MA, USA) and polyvinylidene difluoride membranes (HybondTM ) from GE Healthcare Life Sciences (Pittsburgh, PA, USA).

    Techniques: Activity Assay, Binding Assay, In Vitro, Inhibition, Activation Assay, Incubation, Mutagenesis, Autoradiography

    PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ γ - 32 P]ATP. Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment

    Journal: Cell Death & Disease

    Article Title: SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

    doi: 10.1038/cddis.2014.492

    Figure Lengend Snippet: PKC-dependent phosphorylation of ADAM10 tail does not influence the association to SAP97. ( a ) ADAM10 cytoplasmic tail (Ct) and deletion mutants of aa sequences. In bold is the putative PKC phosphosite. ( b ) GST-ADAM10-Ct and deletion mutants were incubated or not with PKC in the presence of [ γ - 32 P]ATP. Only GST-ADAM10-Ct is phosphorylated. Fusion proteins levels were comparable as shown by Coomassie staining (lower panel). ( c ) In vitro phosphorylation of GST-ADAM10-S741A fusion protein. The mutation S741A leads to a strong reduction of ADAM10-Ct phosphorylation. Fusion protein levels were comparable as shown by Coomassie staining (lower panel). ( d ) After PKC phosphorylation, GST fusion proteins of ADAM10 tail were used to perform pull-down assays to detect SAP97. PKC phosphorylation of ADAM10 does not affect SAP97 binding. ( e ) Quantification of experiments in ( d ) ( n =3, P > 0.05 phosphorylated versus non-phosphorylated, paired t -test). In this and all subsequent pull-down experiments, data were normalized on GST optical density (OD) and shown as the percentage of non-phosphorylated proteins in the same experiment

    Article Snippet: In vitro fusion protein phosphorylation assay GST-purified fusion proteins were incubated or not with 10 ng of active PKC for 30 min at 37 °C, in the presence of 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 0.5 mM CaCl2 and 10 μ M ATP ([γ -32 P]ATP 2 μ Ci per tube; 3000 Ci/mmol; Perkin-Elmer, Waltham, MA, USA).

    Techniques: Incubation, Staining, In Vitro, Mutagenesis, Binding Assay