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PerkinElmer γ 32
Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from  m / z  1507.70 to 1427.75 and  m / z  1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344  and the N-terminal pyroglutamic acid modified DCX 331–344  respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from  m / z  2072.82 to 1992.87 and  m / z  2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39  respectively.
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1) Product Images from "Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5"

Article Title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

Journal: Biochemical Journal

doi: 10.1042/BJ20040324

Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from  m / z  1507.70 to 1427.75 and  m / z  1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344  and the N-terminal pyroglutamic acid modified DCX 331–344  respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from  m / z  2072.82 to 1992.87 and  m / z  2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39  respectively.
Figure Legend Snippet: Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from m / z 1507.70 to 1427.75 and m / z 1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344 and the N-terminal pyroglutamic acid modified DCX 331–344 respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from m / z 2072.82 to 1992.87 and m / z 2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39 respectively.

Techniques Used: SDS Page, High Performance Liquid Chromatography, Concentration Assay, Mass Spectrometry, De-Phosphorylation Assay, Modification

Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.
Figure Legend Snippet: Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.

Techniques Used: Mutagenesis, Recombinant, SDS Page, Autoradiography, Activity Assay

Related Articles

Amplification:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: The primers and conditions for CD3ε amplification are described in ( ). .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′.

Agarose Gel Electrophoresis:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′. .. Blots were then washed, exposed overnight to a phosphoimaging cassette, and visualized using Storm 860 Phosphor Screen and Image Quant (version 5.0) software (Molecular Dynamics, Sunnyvale, CA).

Random Hexamer Labeling:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: Then 1.5 or 5 μg of total RNA were reverse transcribed at 42 C for 50 min using SuperScript II reverse transcriptase (Invitrogen, Burlington, Ontario, Canada) and random hexamer primers (Invitrogen Life Technologies). .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′.

Polymerase Chain Reaction:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′. .. Blots were then washed, exposed overnight to a phosphoimaging cassette, and visualized using Storm 860 Phosphor Screen and Image Quant (version 5.0) software (Molecular Dynamics, Sunnyvale, CA).

Size-exclusion Chromatography:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: These primers result in the amplification of a 1.407-kb product spanning bases 79–1486 of the GLP-1R mRNA sequence. β-Actin cDNA was amplified using the primer pairs 5′-TGACATCCGTAAAGA-3′ and 5′-CAGCTCAGTAACAGTCC-3′, with the following parameters: 94 C, 5 min; 94 C, 30 sec, 45 C, 30 sec, 72 C, 30 sec, 40 cycles; 72 C, 4 min. .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′.

Construct:

Article Title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5
Article Snippet: Paragraph title: Chemicals and DNA constructs ... [γ-32 or 33 P]ATP was from PerkinElmer Life Sciences (Boston, MA, U.S.A.) and trypsin (Gold-MS grade, porcine) was from Promega (Madison, WI, U.S.A.).

Electrophoresis:

Article Title: The Human Lagging Strand DNA Polymerase ? Holoenzyme Is Distributive *
Article Snippet: [α-32 P]ATP, [γ-32 P[ATP, and [α-32 P]dCTP were purchased from PerkinElmer Life Sciences. .. ATPγS was purchased from Calbiochem. dNTPs were from Denville Scientific Inc. Electrophoresis grade agarose was from IBI Scientific.

Article Title: Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index
Article Snippet: After electrophoresis, RIKA gels were washed twice for 30 minutes each with 200 ml 20% isopropanol, 50 mM Tris (pH 7.5) to remove SDS, then with 50 mM Tris (pH 7.5), 2 mM β-mercaptoethanol twice for 30 minutes each at room temperature (18–24 °C). .. In-gel kinase reactions were performed at room temperature in 20 mM Tris (pH 7.5), 20 mM MgCl2 , 2 mM DTT containing 2.5 μCi γ-32 P-ATP/ml (9,000 Ci/mmol; Perkin Elmer) in a total volume of 200 ml.

Concentration Assay:

Article Title: Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index
Article Snippet: In-gel kinase reactions were performed at room temperature in 20 mM Tris (pH 7.5), 20 mM MgCl2 , 2 mM DTT containing 2.5 μCi γ-32 P-ATP/ml (9,000 Ci/mmol; Perkin Elmer) in a total volume of 200 ml. .. CK2 concentration in CK2 RIKA gels was 5 μg/ml, and PKA concentration was 20 μg/ml.

Incubation:

Article Title: Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index
Article Snippet: After incubating in refolding buffer three times for 15 minutes each, followed by overnight incubation at 4 °C, gels were incubated in 200 ml 20 mM Tris (pH 7.5), 20 mM MgCl2 for 30 minutes at room temperature. .. In-gel kinase reactions were performed at room temperature in 20 mM Tris (pH 7.5), 20 mM MgCl2 , 2 mM DTT containing 2.5 μCi γ-32 P-ATP/ml (9,000 Ci/mmol; Perkin Elmer) in a total volume of 200 ml.

Thin Layer Chromatography:

Article Title: The Human Lagging Strand DNA Polymerase ? Holoenzyme Is Distributive *
Article Snippet: [α-32 P]ATP, [γ-32 P[ATP, and [α-32 P]dCTP were purchased from PerkinElmer Life Sciences. .. Polyethyleneimine-modified cellulose (PEI cellulose) TLC plates were obtained from EMD Biosciences.

Labeling:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′. .. Blots were then washed, exposed overnight to a phosphoimaging cassette, and visualized using Storm 860 Phosphor Screen and Image Quant (version 5.0) software (Molecular Dynamics, Sunnyvale, CA).

Expressing:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: Paragraph title: GLP-1R expression ... The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′.

Sequencing:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: These primers result in the amplification of a 1.407-kb product spanning bases 79–1486 of the GLP-1R mRNA sequence. β-Actin cDNA was amplified using the primer pairs 5′-TGACATCCGTAAAGA-3′ and 5′-CAGCTCAGTAACAGTCC-3′, with the following parameters: 94 C, 5 min; 94 C, 30 sec, 45 C, 30 sec, 72 C, 30 sec, 40 cycles; 72 C, 4 min. .. The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′.

Kinase Assay:

Article Title: Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index
Article Snippet: Paragraph title: Reverse in-gel kinase assay ... In-gel kinase reactions were performed at room temperature in 20 mM Tris (pH 7.5), 20 mM MgCl2 , 2 mM DTT containing 2.5 μCi γ-32 P-ATP/ml (9,000 Ci/mmol; Perkin Elmer) in a total volume of 200 ml.

other:

Article Title: Characterization of the Neisseria meningitidis Helicase RecG
Article Snippet: Briefly, oligonucleotides were 5′-end labelled using γ-32 P[ATP] (PerkinElmer) and T4 PNK enzyme (NEB) for 1 h at 37°C.

SDS Page:

Article Title: Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index
Article Snippet: To prepare each 10 ml RIKA SDS-PAGE gel, the following were combined: 3.5 ml kinase (10–1000 μg in 8 M urea, 100 mM NaH2 PO4 (pH 8.0), 250 mM imidazole), 2.5 ml of 0.5 M Tris (pH 8.8), 4 ml 30% acrylamide/bis 37.5:1) (Bio-Rad), 50 μl 10% ammonium persulfate, 50 μl 20% SDS and 10 μl TEMED. .. In-gel kinase reactions were performed at room temperature in 20 mM Tris (pH 7.5), 20 mM MgCl2 , 2 mM DTT containing 2.5 μCi γ-32 P-ATP/ml (9,000 Ci/mmol; Perkin Elmer) in a total volume of 200 ml.

Plasmid Preparation:

Article Title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5
Article Snippet: [γ-32 or 33 P]ATP was from PerkinElmer Life Sciences (Boston, MA, U.S.A.) and trypsin (Gold-MS grade, porcine) was from Promega (Madison, WI, U.S.A.). .. GST (glutathione S -transferase)-cdk5 plasmid was from J. Wang (Hong Kong University, Hong Kong) and the GST-p25 plasmid was from L.-H. Tsai (Harvard, U.S.A.) as we have described previously [ ].

Software:

Article Title: Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice
Article Snippet: The PCR products were electrophoresed in a 1% agarose gel, transferred to a Nytran Super Charge nylon membrane (Mandel Scientific, Guelph, Ontario, Canada), and hybridized overnight using the following internal primers labeled by T4 polynucleotide kinase reaction with γ-32 P[ATP] (PerkinElmer, Wellesley, MA): GLP-1R 5′-GCTGTATCTGAGCATAGGCT-3′; β-actin 5′-GATCATTGCTCCTCCTGAGC-3′; CD3ε 5′-GAGCACCCTGCTACTCCTTG-3′. .. Blots were then washed, exposed overnight to a phosphoimaging cassette, and visualized using Storm 860 Phosphor Screen and Image Quant (version 5.0) software (Molecular Dynamics, Sunnyvale, CA).

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    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, 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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    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Journal: Molecular Biology of the Cell

    Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

    doi: 10.1091/mbc.E10-07-0636

    Figure Lengend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

    Article Snippet: In Vitro Kinase Assays pET and pET-Aly2 were incubated for 30 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 , 1 mM DTT, 1 μM unlabeled ATP, aprotinin, and leupeptin) with 75 nM [γ-32 P]ATP (Perkin Elmer-Cetus) with or without Npr1 kinase (purified from Y258 yeast cells).

    Techniques: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

    Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .

    Journal: PLoS ONE

    Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    doi: 10.1371/journal.pone.0124058

    Figure Lengend Snippet: Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .

    Article Snippet: Phosphotransfer assays Purified LOVHK protein at a concentration of 2.5 μM was irradiated with 10 flashes of white light (a fluence of 4000 umol m-2 sec-1 of a xenon flashlamp) in phosphorylation buffer (20 mM Tris-HCl pH 8, 50 mM NaCl, 5 mM MgCl2 , 100 μM ATP) containing 10 μCi of [γ-32 P] ATP (111TBq/mmol, PerkinElmer Life Sciences).

    Techniques: Purification, Concentration Assay, SDS Page