Structured Review

GE Healthcare γ 32 p atp
Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 <t>P]ATP</t> and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.
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Images

1) Product Images from "ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells"

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.7.2204-2219.2002

Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.
Figure Legend Snippet: Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

Techniques Used: Activity Assay, Over Expression, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, SDS Page, Cell Culture, Expressing

2) Product Images from "Analysis of p53-RNA Interactions in Cultured Human Cells"

Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2007.08.181

RNA is purified from cell lysates by anti-p53 antibodies regardless of p53 status. (A) Western analysis of p53. Preparations of whole cell lysate (WCL; 10% of total) [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4, after IP with anti-p53 Do-7 (lanes 5–8, respectively). (B) Co-immunoprecipitated RNA after [γ 32 P]-ATP labeling: HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4. Formaldehyde cross-linked whole cell lysate: [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4], subjected to IP with anti-p53 Do-7 followed by RNA radiolabeling. (C) Western blot with anti-p53 antibody Do-1. (D) Membrane from panel (C) exposed to film.
Figure Legend Snippet: RNA is purified from cell lysates by anti-p53 antibodies regardless of p53 status. (A) Western analysis of p53. Preparations of whole cell lysate (WCL; 10% of total) [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4, after IP with anti-p53 Do-7 (lanes 5–8, respectively). (B) Co-immunoprecipitated RNA after [γ 32 P]-ATP labeling: HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4. Formaldehyde cross-linked whole cell lysate: [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4], subjected to IP with anti-p53 Do-7 followed by RNA radiolabeling. (C) Western blot with anti-p53 antibody Do-1. (D) Membrane from panel (C) exposed to film.

Techniques Used: Purification, Western Blot, Immunoprecipitation, Labeling, Radioactivity

3) Product Images from "The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus"

Article Title: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2012.08015.x

In vitro phosphorylation of SgmT and DigR. A. Schematic representation of the domain organization of SgmT and DigR. Constructs used in in vitro phosphorylation and c-di-GMP binding experiments are indicated. Numbers indicate the co-ordinates of the constructs with respect to the full-length SgmT. B. Autophosphorylation of SgmT kinase and phosphotransfer to DigR or SgmT receiver . SgmT kinase (10 µM final concentration) was incubated in the presence of 1.0 mM [γ- 32 P]-ATP at 30°C for 30 min and then the reaction was either stopped or DigR or SgmT receiver added (10 µM final concentration) for 2 min and 5 min respectively. Samples were separated by SDS-PAGE without prior heating and detected by phosphorimaging. C. Autophosphorylation of SgmT kinase receiver and phosphotransfer to DigR. Samples were treated as in (B). D. Autophosphorylation of full-length SgmT and phosphotransfer to DigR. Samples were treated as described in (B). The asterisk (*) indicates a background band. E. c-di-GMP does not affect full-length SgmT autophosphorylation or phosphotransfer to DigR. Samples were treated as described in (B) except that c-di-GMP was added in the indicated concentrations during autophosphorylation and phosphotransfer. The asterisk (*) indicates a background band.
Figure Legend Snippet: In vitro phosphorylation of SgmT and DigR. A. Schematic representation of the domain organization of SgmT and DigR. Constructs used in in vitro phosphorylation and c-di-GMP binding experiments are indicated. Numbers indicate the co-ordinates of the constructs with respect to the full-length SgmT. B. Autophosphorylation of SgmT kinase and phosphotransfer to DigR or SgmT receiver . SgmT kinase (10 µM final concentration) was incubated in the presence of 1.0 mM [γ- 32 P]-ATP at 30°C for 30 min and then the reaction was either stopped or DigR or SgmT receiver added (10 µM final concentration) for 2 min and 5 min respectively. Samples were separated by SDS-PAGE without prior heating and detected by phosphorimaging. C. Autophosphorylation of SgmT kinase receiver and phosphotransfer to DigR. Samples were treated as in (B). D. Autophosphorylation of full-length SgmT and phosphotransfer to DigR. Samples were treated as described in (B). The asterisk (*) indicates a background band. E. c-di-GMP does not affect full-length SgmT autophosphorylation or phosphotransfer to DigR. Samples were treated as described in (B) except that c-di-GMP was added in the indicated concentrations during autophosphorylation and phosphotransfer. The asterisk (*) indicates a background band.

Techniques Used: In Vitro, Construct, Binding Assay, Concentration Assay, Incubation, SDS Page

4) Product Images from "p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *"

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.123497

PAK4 phosphorylates the integrin β5 subunit. PAK4 was immunoprecipitated using an anti-HA mAb from COS-7 cells transfected with an HA-PAK4 vector and incubated with integrins in the presence of [γ- 32 P]ATP. A , PAK4 phosphorylation of integrin
Figure Legend Snippet: PAK4 phosphorylates the integrin β5 subunit. PAK4 was immunoprecipitated using an anti-HA mAb from COS-7 cells transfected with an HA-PAK4 vector and incubated with integrins in the presence of [γ- 32 P]ATP. A , PAK4 phosphorylation of integrin

Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Incubation

5) Product Images from "Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization"

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.3.852-863.2003

S6KβII, but not S6KαII, is phosphorylated at the C terminus by different PKC isoforms in vitro. (A) Schematic representation of S6KβI and S6KβII and their deletion mutants, which lack amino- and carboxyl-terminal sequences. Major domain boundaries are indicated. Structural features are indicated as follows: grey boxes indicate unique proline-rich sequences of S6Kβ; solid black boxes indicate NLSs (NLS1 and NLS2); striped boxes correspond to potential NESs. The N- and C-terminal amino acid sequences, containing NES and NLS, are shown above the diagrams. All recombinant constructs carry an N-terminal EE-tag sequence, and deleted amino acids are indicated. (B) In vitro phosphorylation of bacterially expressed His-S6KαC and His-S6KβC by various PKCs. Affinity-purified His-tagged S6Kα and S6Kβ C-terminal peptides were incubated in the presence of different recombinant PKC isoforms and [γ- 32 P]ATP. The reaction mixtures were separated by SDS-PAGE and stained with Coomassie. The dried gel was analyzed by autoradiography. (C) In vitro phosphorylation of recombinant full-length S6KαII, S6KβII, and deleted S6KβII mutants by PKCs. HEK 293 cells transiently transfected with wild-type EE-S6KαII, EE-S6KβII, EE-S6KβIIΔN, or EE-S6KβIIΔC were serum starved for 24 h, and recombinant proteins were immunoprecipitated with anti-EE-tag antibody. The immunoprecipitates were incubated with [γ- 32 P]ATP in the absence or presence of different recombinant PKC isoforms. The reaction mixtures were analyzed as described above.
Figure Legend Snippet: S6KβII, but not S6KαII, is phosphorylated at the C terminus by different PKC isoforms in vitro. (A) Schematic representation of S6KβI and S6KβII and their deletion mutants, which lack amino- and carboxyl-terminal sequences. Major domain boundaries are indicated. Structural features are indicated as follows: grey boxes indicate unique proline-rich sequences of S6Kβ; solid black boxes indicate NLSs (NLS1 and NLS2); striped boxes correspond to potential NESs. The N- and C-terminal amino acid sequences, containing NES and NLS, are shown above the diagrams. All recombinant constructs carry an N-terminal EE-tag sequence, and deleted amino acids are indicated. (B) In vitro phosphorylation of bacterially expressed His-S6KαC and His-S6KβC by various PKCs. Affinity-purified His-tagged S6Kα and S6Kβ C-terminal peptides were incubated in the presence of different recombinant PKC isoforms and [γ- 32 P]ATP. The reaction mixtures were separated by SDS-PAGE and stained with Coomassie. The dried gel was analyzed by autoradiography. (C) In vitro phosphorylation of recombinant full-length S6KαII, S6KβII, and deleted S6KβII mutants by PKCs. HEK 293 cells transiently transfected with wild-type EE-S6KαII, EE-S6KβII, EE-S6KβIIΔN, or EE-S6KβIIΔC were serum starved for 24 h, and recombinant proteins were immunoprecipitated with anti-EE-tag antibody. The immunoprecipitates were incubated with [γ- 32 P]ATP in the absence or presence of different recombinant PKC isoforms. The reaction mixtures were analyzed as described above.

Techniques Used: In Vitro, Recombinant, Construct, Sequencing, Affinity Purification, Incubation, SDS Page, Staining, Autoradiography, Transfection, Immunoprecipitation

Identification of PKC phosphorylation site and characterization of phosphospecific S6Kβ antibody. (A) Mass spectroscopy analysis of PKC phosphorylation site in S6KβII. The amino acid sequence of His-S6KβC is shown on top. (B and C) Analysis of specificity of anti-pS486 antibody. Bacterially expressed His-S6KβC was incubated with [γ- 32 P]ATP in the presence (+) or absence −) of recombinant PKCβII. Samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by autoradiography (B) or immunoblotting with anti-pS486 antibody (C). HI, histone H1; PVDF, polyvinylidene difluoride; WB, Western blot.
Figure Legend Snippet: Identification of PKC phosphorylation site and characterization of phosphospecific S6Kβ antibody. (A) Mass spectroscopy analysis of PKC phosphorylation site in S6KβII. The amino acid sequence of His-S6KβC is shown on top. (B and C) Analysis of specificity of anti-pS486 antibody. Bacterially expressed His-S6KβC was incubated with [γ- 32 P]ATP in the presence (+) or absence −) of recombinant PKCβII. Samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by autoradiography (B) or immunoblotting with anti-pS486 antibody (C). HI, histone H1; PVDF, polyvinylidene difluoride; WB, Western blot.

Techniques Used: Mass Spectrometry, Sequencing, Incubation, Recombinant, SDS Page, Autoradiography, Western Blot

6) Product Images from "The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †"

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.23.8718-8728.2003

Cak1p is required for the catalytic activity of Ime2p. Epitope-tagged Ime2p was immunoprecipitated (IP) from sporulating CAK1 (KSY137) or cak1- Δ (KSY138) diploid cells in the CDC28-43244 background at the indicated times. Protein kinase activity was measured by incubation of Ime2p-myc with purified MBP-Ndt80p substrate and [γ- 32 P]ATP. *P-Ime2 and *P-Ndt80 correspond to Ime2p autophosphorylation and phosphorylation of Ndt80p, respectively. Immunoblot analysis shows that comparable levels of Ime2p-myc are present in the reaction mixtures.
Figure Legend Snippet: Cak1p is required for the catalytic activity of Ime2p. Epitope-tagged Ime2p was immunoprecipitated (IP) from sporulating CAK1 (KSY137) or cak1- Δ (KSY138) diploid cells in the CDC28-43244 background at the indicated times. Protein kinase activity was measured by incubation of Ime2p-myc with purified MBP-Ndt80p substrate and [γ- 32 P]ATP. *P-Ime2 and *P-Ndt80 correspond to Ime2p autophosphorylation and phosphorylation of Ndt80p, respectively. Immunoblot analysis shows that comparable levels of Ime2p-myc are present in the reaction mixtures.

Techniques Used: Activity Assay, Immunoprecipitation, Incubation, Purification

7) Product Images from "Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction"

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.01765

Potential activity and sites of Cdk2/Cyclin phosphorylation on Ebola VP40. (A) The amino acid sequence of Ebola VP40 with potential phosphorylation sites ( ∗ ) and hypothesized Cdk2 phosphorylation site (red). (B) Crystal structure of EBOV VP40 monomer. Potential phosphorylation sites are marked on two serine residues (red) and two threonine residues (blue). Previously demonstrated c-Abl1 phosphorylation site tyrosine-13 ( García et al., 2012 ) is indicated (green). The Ebola VP40 crystal structure modified was from Dessen et al. (2000) . (C) CEM extracts were used for immunoprecipitation using α-Cdk2, α-Cyclin E, α-Cyclin A or IgG as control. Five hundred micrograms of CEM whole cell extract was used with 10 μg of each antibody and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C, followed by next day addition of Protein A/G for 2 h at 4°C. The complexes were washed with twice with TNE50 + 0.1% NP-40 and twice with kinase buffer, and successively used for in vitro kinase assay usi ng purified VP40 protein (lanes 3–9) and [γ- 32 P] ATP. Cdk inhibitors Alsterpaullone, Indirubin-3′-monoxime, Purvalanol A and r-Roscovitine (lanes 6–9) were used at final 1 μM concentration for 1 h at 37°C. Densitometry analysis of kinase activity as a percentage is shown in the bottom panel with α-Cdk2+VP40 set to 100%. (D) 293T cells were transfected with 20 μg of VP40 DNA using electroporation and were placed under antibiotic selection (Hygromycin B). After 4 days, cells (30–40% confluent) were either untreated or blocked at G1/S phase of cell cycle with Hydroxyurea (1 mM) for 1 day. Cells were subsequently radio-labeled with [γ- 32 P] ATP and a few samples were treated with r-Roscovitine (10 μM) for 4 h in complete media. Radioactive material was subsequently removed, washed and chased with complete media for 2 h. Next, cells were removed (cell scraper), lysed with lysis buffer, and immunoprecipitation was performed overnight with α-VP40 antibody in TNE150 + 0.1% NP-40 buffer. Protein A/G was added for 2 h and complexes were washed twice with TNE150 + 0.1% NP-40 and once with kinase buffer. Radioactive immunoprecipitated complexes were resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, dried, and exposed to phosphoroImager cassette. Densitometry analysis of kinase activity as determined by ImageJ software is shown as a percentage in the bottom panel, with untreated VP40-transfected 293T cells set to 100%. (E) Five hundred micrograms of CEM whole cell extract was immunoprecipitated with 10 μg of α-Cyclin E or normal rabbit IgG antibody in TNE50 + 0.1% NP-40, and incubated overnight at 4°C. Fifty microliters of a 30% slurry of Protein A/G was added next day, incubated for 2 h, washed twice with PBS and once with kinase buffer, and then resuspended in kinase buffer. Fifty microgram of 10–12 mer peptides matching potential phosphorylation sites on EBOV VP40 were added to 15 μL of sample IP and 2 μL of a [γ- 32 P] ATP and kinase buffer solution (1:3). Samples were incubated for 1 h before being dotted onto Whatman glass microfibre filters and dried for 30 min before being submerged in 1x TE buffer with gentle agitation for 2 days. DPM2 counts were then taken with a scintillation counter (QuantaSmart TM ). The peptide sequences used are illustrated in the boxes. Underlined letters indicate the potentially phosphorylated residue. Residues that were altered from the wild type sequence are indicated (red).
Figure Legend Snippet: Potential activity and sites of Cdk2/Cyclin phosphorylation on Ebola VP40. (A) The amino acid sequence of Ebola VP40 with potential phosphorylation sites ( ∗ ) and hypothesized Cdk2 phosphorylation site (red). (B) Crystal structure of EBOV VP40 monomer. Potential phosphorylation sites are marked on two serine residues (red) and two threonine residues (blue). Previously demonstrated c-Abl1 phosphorylation site tyrosine-13 ( García et al., 2012 ) is indicated (green). The Ebola VP40 crystal structure modified was from Dessen et al. (2000) . (C) CEM extracts were used for immunoprecipitation using α-Cdk2, α-Cyclin E, α-Cyclin A or IgG as control. Five hundred micrograms of CEM whole cell extract was used with 10 μg of each antibody and 100 μL TNE50 + 0.1% NP-40 for 48 h at 4°C, followed by next day addition of Protein A/G for 2 h at 4°C. The complexes were washed with twice with TNE50 + 0.1% NP-40 and twice with kinase buffer, and successively used for in vitro kinase assay usi ng purified VP40 protein (lanes 3–9) and [γ- 32 P] ATP. Cdk inhibitors Alsterpaullone, Indirubin-3′-monoxime, Purvalanol A and r-Roscovitine (lanes 6–9) were used at final 1 μM concentration for 1 h at 37°C. Densitometry analysis of kinase activity as a percentage is shown in the bottom panel with α-Cdk2+VP40 set to 100%. (D) 293T cells were transfected with 20 μg of VP40 DNA using electroporation and were placed under antibiotic selection (Hygromycin B). After 4 days, cells (30–40% confluent) were either untreated or blocked at G1/S phase of cell cycle with Hydroxyurea (1 mM) for 1 day. Cells were subsequently radio-labeled with [γ- 32 P] ATP and a few samples were treated with r-Roscovitine (10 μM) for 4 h in complete media. Radioactive material was subsequently removed, washed and chased with complete media for 2 h. Next, cells were removed (cell scraper), lysed with lysis buffer, and immunoprecipitation was performed overnight with α-VP40 antibody in TNE150 + 0.1% NP-40 buffer. Protein A/G was added for 2 h and complexes were washed twice with TNE150 + 0.1% NP-40 and once with kinase buffer. Radioactive immunoprecipitated complexes were resuspended in Laemmli buffer, run on a 4–20% Tris-glycine SDS gel, dried, and exposed to phosphoroImager cassette. Densitometry analysis of kinase activity as determined by ImageJ software is shown as a percentage in the bottom panel, with untreated VP40-transfected 293T cells set to 100%. (E) Five hundred micrograms of CEM whole cell extract was immunoprecipitated with 10 μg of α-Cyclin E or normal rabbit IgG antibody in TNE50 + 0.1% NP-40, and incubated overnight at 4°C. Fifty microliters of a 30% slurry of Protein A/G was added next day, incubated for 2 h, washed twice with PBS and once with kinase buffer, and then resuspended in kinase buffer. Fifty microgram of 10–12 mer peptides matching potential phosphorylation sites on EBOV VP40 were added to 15 μL of sample IP and 2 μL of a [γ- 32 P] ATP and kinase buffer solution (1:3). Samples were incubated for 1 h before being dotted onto Whatman glass microfibre filters and dried for 30 min before being submerged in 1x TE buffer with gentle agitation for 2 days. DPM2 counts were then taken with a scintillation counter (QuantaSmart TM ). The peptide sequences used are illustrated in the boxes. Underlined letters indicate the potentially phosphorylated residue. Residues that were altered from the wild type sequence are indicated (red).

Techniques Used: Activity Assay, Sequencing, Modification, Immunoprecipitation, In Vitro, Kinase Assay, Purification, Concentration Assay, Transfection, Electroporation, Selection, Labeling, Lysis, SDS-Gel, Software, Incubation

8) Product Images from "Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞"

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M809724200

PP1 analogs specifically inhibit Pto as kinase activity. A, in vitro phosphorylation of the kinase-deficient mutant GST-Pti1(K96N) by GST-Pto ( white bars ) and GST-Pto as ( gray bars ) in the presence of [γ- 32 P]ATP and the indicated PP1 analogs
Figure Legend Snippet: PP1 analogs specifically inhibit Pto as kinase activity. A, in vitro phosphorylation of the kinase-deficient mutant GST-Pti1(K96N) by GST-Pto ( white bars ) and GST-Pto as ( gray bars ) in the presence of [γ- 32 P]ATP and the indicated PP1 analogs

Techniques Used: Activity Assay, In Vitro, Mutagenesis

9) Product Images from "IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION"

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

Journal: Nucleosides, nucleotides & nucleic acids

doi: 10.1080/15257770802257903

Minimization of clone 4–15 cellulose-binding RNA. (A) Locations of a series of successive 10 nucleotide truncations (TR1 through TR6) used to assess the minimal length of the clone 4–15 aptamer that retains function. Shaded nucleotides reflect the minimal functional RNA derived from the data depicted in Fig. 3C. (B) Paper chromatogram assessing the cellulose-binding functions of various 32 P-labeled RNA constructs. Random-sequence G0 RNAs are used as a control. (C) Single-nucleotide-resolution mapping of boundaries of the cellulose-binding RNA aptamer 4–15. RNAs were labeled on 5′ or 3′ termini using [γ- 32 P]ATP or [ 32 P]pCp, respectively. Radiolabeled RNAs were subjected to partial digestion with alkali and then were applied to cellulose-packed columns. RNA fragments retained by the cellulose matrix were recovered using elution buffer (lanes 1 and 3). RNAs that were unable to bind to cellulose were also collected (lanes 2 and 4).
Figure Legend Snippet: Minimization of clone 4–15 cellulose-binding RNA. (A) Locations of a series of successive 10 nucleotide truncations (TR1 through TR6) used to assess the minimal length of the clone 4–15 aptamer that retains function. Shaded nucleotides reflect the minimal functional RNA derived from the data depicted in Fig. 3C. (B) Paper chromatogram assessing the cellulose-binding functions of various 32 P-labeled RNA constructs. Random-sequence G0 RNAs are used as a control. (C) Single-nucleotide-resolution mapping of boundaries of the cellulose-binding RNA aptamer 4–15. RNAs were labeled on 5′ or 3′ termini using [γ- 32 P]ATP or [ 32 P]pCp, respectively. Radiolabeled RNAs were subjected to partial digestion with alkali and then were applied to cellulose-packed columns. RNA fragments retained by the cellulose matrix were recovered using elution buffer (lanes 1 and 3). RNAs that were unable to bind to cellulose were also collected (lanes 2 and 4).

Techniques Used: Binding Assay, Functional Assay, Derivative Assay, Labeling, Construct, Sequencing

10) Product Images from "Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3"

Article Title: Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3

Journal: Biochemical Journal

doi: 10.1042/BJ20041057

Identification of 45 kDa and 35 kDa proteins that are phosphor- ylated by SGK1, but only weakly by PKBα The proteins not retained on heparin–Sepharose were chromatographed on Source 15 Q-Sepharose, as described in the Materials and methods section. Each fraction was incubated for 4 min at 30 °C with 2 mM MgCl 2 /20 nM [γ- 32 P]ATP in the absence (−) or presence (+) of 0.3 unit/ml SGK1 or PKBα, denatured in SDS, subjected to SDS/PAGE, transferred to ImmobilonP membranes and autoradiographed. Two substrates for SGK1 with apparent molecular masses of 45 kDa and 35 kDa, eluting between 0.11 M and 0.22 M NaCl, were detected by autoradiography.
Figure Legend Snippet: Identification of 45 kDa and 35 kDa proteins that are phosphor- ylated by SGK1, but only weakly by PKBα The proteins not retained on heparin–Sepharose were chromatographed on Source 15 Q-Sepharose, as described in the Materials and methods section. Each fraction was incubated for 4 min at 30 °C with 2 mM MgCl 2 /20 nM [γ- 32 P]ATP in the absence (−) or presence (+) of 0.3 unit/ml SGK1 or PKBα, denatured in SDS, subjected to SDS/PAGE, transferred to ImmobilonP membranes and autoradiographed. Two substrates for SGK1 with apparent molecular masses of 45 kDa and 35 kDa, eluting between 0.11 M and 0.22 M NaCl, were detected by autoradiography.

Techniques Used: Incubation, SDS Page, Autoradiography

11) Product Images from "Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-? and -?"

Article Title: Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-? and -?

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.01-07-0323

(A) Na,K-ATPase activity, as measured by [γ- 32 P]ATP hydrolysis, in AT2 cells incubated for 15 min at room temperature with 1 μM DA, FEN, or quinpirole (QP). Each bar represents the mean ± SEM of four determinations performed independently (separate cell isolations) and in triplicate. ∗p
Figure Legend Snippet: (A) Na,K-ATPase activity, as measured by [γ- 32 P]ATP hydrolysis, in AT2 cells incubated for 15 min at room temperature with 1 μM DA, FEN, or quinpirole (QP). Each bar represents the mean ± SEM of four determinations performed independently (separate cell isolations) and in triplicate. ∗p

Techniques Used: Activity Assay, Incubation

12) Product Images from "Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA"

Article Title: Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA

Journal: Nucleic Acids Research

doi:

DNA circularisation by LrpC. The 182 bp [γ- 32 P]DNA fragment (0.5 nM) was incubated with increasing concentrations of the LrpC protein (30–500 nM, lanes 2–6 and 8–12) in buffer C containing 50 mM NaCl and 1 mM ATP for 15 min at 37°C. In samples 1–6 the DNA was further incubated with ligase buffer and in lanes 7–12 with 1 U of T4 DNA ligase for 30 min at 16°C. The samples were deproteinised and analysed by 4% non-denaturing PAGE in 0.5× TBE, and autoradiographs of the dried gels were subsequently taken.
Figure Legend Snippet: DNA circularisation by LrpC. The 182 bp [γ- 32 P]DNA fragment (0.5 nM) was incubated with increasing concentrations of the LrpC protein (30–500 nM, lanes 2–6 and 8–12) in buffer C containing 50 mM NaCl and 1 mM ATP for 15 min at 37°C. In samples 1–6 the DNA was further incubated with ligase buffer and in lanes 7–12 with 1 U of T4 DNA ligase for 30 min at 16°C. The samples were deproteinised and analysed by 4% non-denaturing PAGE in 0.5× TBE, and autoradiographs of the dried gels were subsequently taken.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

13) Product Images from "Convergence of TOR-Nitrogen and Snf1-Glucose Signaling Pathways onto Gln3"

Article Title: Convergence of TOR-Nitrogen and Snf1-Glucose Signaling Pathways onto Gln3

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.4.1246-1252.2002

Snf1 interacts with Gln3. (A) Snf1 interacts with Gln3 in a yeast two-hybrid assay. Gal4 DNA binding domain (BD)-Snf1 specifically interacts with Gal4 activation domain (AD)-Gln3, but not with AD alone. The two-hybrid reporter strain (AH109) carrying different BD and AD fusion plasmids was assayed for growth on an adenine dropout plate or a histidine dropout plate containing 3-AT (2 mM). LgT, large T antigen. (B) Snf1 binds to GST-Gln3. Extracts of wild-type (WT) and snf1 Δ yeast strains were assayed for their ability to bind to immobilized, bacterially produced GST or GST-Gln3. The bound materials were detected by Western blotting with an anti-polyhistidine antibody that recognizes Snf1. (C) Snf1 phosphorylates Gln3 in vitro. HA-Snf1 was immunoprecipitated and assayed for its ability to phosphorylate bacterial recombinant GST-Gln3 in the presence of [γ- 32 P]ATP.
Figure Legend Snippet: Snf1 interacts with Gln3. (A) Snf1 interacts with Gln3 in a yeast two-hybrid assay. Gal4 DNA binding domain (BD)-Snf1 specifically interacts with Gal4 activation domain (AD)-Gln3, but not with AD alone. The two-hybrid reporter strain (AH109) carrying different BD and AD fusion plasmids was assayed for growth on an adenine dropout plate or a histidine dropout plate containing 3-AT (2 mM). LgT, large T antigen. (B) Snf1 binds to GST-Gln3. Extracts of wild-type (WT) and snf1 Δ yeast strains were assayed for their ability to bind to immobilized, bacterially produced GST or GST-Gln3. The bound materials were detected by Western blotting with an anti-polyhistidine antibody that recognizes Snf1. (C) Snf1 phosphorylates Gln3 in vitro. HA-Snf1 was immunoprecipitated and assayed for its ability to phosphorylate bacterial recombinant GST-Gln3 in the presence of [γ- 32 P]ATP.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Produced, Western Blot, In Vitro, Immunoprecipitation, Recombinant

14) Product Images from "Saccharomyces cerevisiae Msh2p and Msh6p ATPase Activities Are Both Required during Mismatch Repair"

Article Title: Saccharomyces cerevisiae Msh2p and Msh6p ATPase Activities Are Both Required during Mismatch Repair

Journal: Molecular and Cellular Biology

doi:

(A) ATP hydrolysis activity exhibited by wild-type and mutant Msh2p-Msh6p complexes. Fraction IV of Msh2p-Msh6p, msh2-GD693p-Msh6p, Msh2p-msh6-GD987p, and msh2-GD693p-msh6-GD987p preparations (0.3 μg in each case) was incubated in the presence of 1.2 to 33.3 μM [γ- 32 P]ATP, and the rate of ATP hydrolysis (V) was determined for duplicate reactions after a 15-min incubation at 30°C (Materials and Methods). The results from duplicates were averaged, and the range between the two values is shown. (B) Comparison of ATP hydrolysis activities for wild-type and mutant Msh2p-Msh6p complexes in the presence of homoduplex and mismatch substrates. Msh2p-Msh6p, msh2-GD693p-Msh6p, and Msh2p-msh6-GD987p complexes (0.3 μg of each) were incubated with 33.3 μM [γ- 32 P]ATP and 167 nM each indicated +1 or homoduplex (hom) substrate. The rate of ATP hydrolysis (V) was determined after a 15-min incubation at 30°C. The results from duplicate reactions were averaged, and the range between the two values is shown.
Figure Legend Snippet: (A) ATP hydrolysis activity exhibited by wild-type and mutant Msh2p-Msh6p complexes. Fraction IV of Msh2p-Msh6p, msh2-GD693p-Msh6p, Msh2p-msh6-GD987p, and msh2-GD693p-msh6-GD987p preparations (0.3 μg in each case) was incubated in the presence of 1.2 to 33.3 μM [γ- 32 P]ATP, and the rate of ATP hydrolysis (V) was determined for duplicate reactions after a 15-min incubation at 30°C (Materials and Methods). The results from duplicates were averaged, and the range between the two values is shown. (B) Comparison of ATP hydrolysis activities for wild-type and mutant Msh2p-Msh6p complexes in the presence of homoduplex and mismatch substrates. Msh2p-Msh6p, msh2-GD693p-Msh6p, and Msh2p-msh6-GD987p complexes (0.3 μg of each) were incubated with 33.3 μM [γ- 32 P]ATP and 167 nM each indicated +1 or homoduplex (hom) substrate. The rate of ATP hydrolysis (V) was determined after a 15-min incubation at 30°C. The results from duplicate reactions were averaged, and the range between the two values is shown.

Techniques Used: Activity Assay, Mutagenesis, Incubation

15) Product Images from "The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta"

Article Title: The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Recombinant SRK autophosphorylates on serine and threonine residues in a membranous environment. ( A ) Microsomes from uninfected Sf 21 cells (C) or from Sf 21 cells expressing SRK 3 HA (HA), SRK 3 His (His) or kinase defective m SRK 3 His ( m His) were radiolabeled with [γ- 32 P]ATP. In some cases, proteins (lanes 4 and 5) were purified on Ni-NTA agarose beads after radiolabeling. Proteins were separated by SDS/PAGE and detected by autoradiography. ( B ) Radiolabeled SRK 3 His was purified on Ni-NTA agarose beads and hydrolyzed. Free amino acids were separated in two dimensions by chromatography and electrophoresis as indicated, and radiolabeled amino acids were detected by autoradiography. Phosphoamino acids (P-ser, P-thr, and P-tyr for phosphoserine, phosphothreonine, and phosphotyrosine, respectively) were positioned by staining nonradiolabeled phosphoamino acids, added before separation, with nihydrin. Pi indicates inorganic phosphate.
Figure Legend Snippet: Recombinant SRK autophosphorylates on serine and threonine residues in a membranous environment. ( A ) Microsomes from uninfected Sf 21 cells (C) or from Sf 21 cells expressing SRK 3 HA (HA), SRK 3 His (His) or kinase defective m SRK 3 His ( m His) were radiolabeled with [γ- 32 P]ATP. In some cases, proteins (lanes 4 and 5) were purified on Ni-NTA agarose beads after radiolabeling. Proteins were separated by SDS/PAGE and detected by autoradiography. ( B ) Radiolabeled SRK 3 His was purified on Ni-NTA agarose beads and hydrolyzed. Free amino acids were separated in two dimensions by chromatography and electrophoresis as indicated, and radiolabeled amino acids were detected by autoradiography. Phosphoamino acids (P-ser, P-thr, and P-tyr for phosphoserine, phosphothreonine, and phosphotyrosine, respectively) were positioned by staining nonradiolabeled phosphoamino acids, added before separation, with nihydrin. Pi indicates inorganic phosphate.

Techniques Used: Recombinant, Expressing, Purification, Radioactivity, SDS Page, Autoradiography, Chromatography, Electrophoresis, Staining

16) Product Images from "Concurrent Replication and Methylation at Mammalian Origins of Replication"

Article Title: Concurrent Replication and Methylation at Mammalian Origins of Replication

Journal: Molecular and Cellular Biology

doi:

(A) Fractionation of nascent DNA from human normal skin fibroblasts. DNA was prepared and extruded by branch migration as described in Materials and Methods from human normal skin fibroblasts and size fractionated on a sucrose gradient. The fractions were electrophoresed on a 1% agarose gel and ethidium bromide stained. Okazaki fragments (fractions 1 to 4) as well as higher-molecular-weight nascent DNA (fractions 8 to 10) were used as substrates for nearest-neighbor analysis Lane 1 (M), a 100-bp ladder marker; lanes 2 to 14, nascent DNA fractions with increasing molecular weights. (B) Fractions 1 to 4 containing mostly Okazaki fragments. DNA from fractions 1 to 4 was 5′ labeled with [γ- 32 P]ATP with PNK and subjected to alkaline treatment with 0.4 M NaOH. The alkaline-treated and untreated samples were electrophoresed through a 5% polyacrylamide alkaline gel. An autoradiogram of the dried gel is shown. The lability of the 5′ label in NaOH suggests that most of the DNAs in these fractions bear RNA nucleotides as expected from Okazaki fragments.
Figure Legend Snippet: (A) Fractionation of nascent DNA from human normal skin fibroblasts. DNA was prepared and extruded by branch migration as described in Materials and Methods from human normal skin fibroblasts and size fractionated on a sucrose gradient. The fractions were electrophoresed on a 1% agarose gel and ethidium bromide stained. Okazaki fragments (fractions 1 to 4) as well as higher-molecular-weight nascent DNA (fractions 8 to 10) were used as substrates for nearest-neighbor analysis Lane 1 (M), a 100-bp ladder marker; lanes 2 to 14, nascent DNA fractions with increasing molecular weights. (B) Fractions 1 to 4 containing mostly Okazaki fragments. DNA from fractions 1 to 4 was 5′ labeled with [γ- 32 P]ATP with PNK and subjected to alkaline treatment with 0.4 M NaOH. The alkaline-treated and untreated samples were electrophoresed through a 5% polyacrylamide alkaline gel. An autoradiogram of the dried gel is shown. The lability of the 5′ label in NaOH suggests that most of the DNAs in these fractions bear RNA nucleotides as expected from Okazaki fragments.

Techniques Used: Fractionation, Migration, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker, Labeling

17) Product Images from "Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function"

Article Title: Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function

Journal: Journal of Virology

doi: 10.1128/JVI.78.7.3542-3552.2004

(A) Physical association of EBNA-2 with p34 cdc2 kinase. Whole cell X50-7 lysates from asynchronous or nocodazole-arrested M-phase cells (A and M, respectively) were immunoprecipitated with p34 cdc2 antibody or normal mouse IgG; immune complexes were separated by electrophoresis. EBNA-2 and p34 cdc2 were detected by immunoblotting. (B) Temporal correlation between EBNA-2 hyperphosphorylation and p34 cdc2 kinase activity. X50-7 cells were continuously treated with nocodazole for the indicated times, and whole-cell lysates were separated by electrophoresis and immunoblotted with EBNA-2 antibody (upper panel). For the kinase assay, p34 cdc2 /cyclin B1-kinase complex was immunoprecipitated from the same cell lysates, and kinase activity was determined by [γ- 32 p]ATP incorporation into histone H1 (middle panel) and measured with a PhosphorImager (lower panel).
Figure Legend Snippet: (A) Physical association of EBNA-2 with p34 cdc2 kinase. Whole cell X50-7 lysates from asynchronous or nocodazole-arrested M-phase cells (A and M, respectively) were immunoprecipitated with p34 cdc2 antibody or normal mouse IgG; immune complexes were separated by electrophoresis. EBNA-2 and p34 cdc2 were detected by immunoblotting. (B) Temporal correlation between EBNA-2 hyperphosphorylation and p34 cdc2 kinase activity. X50-7 cells were continuously treated with nocodazole for the indicated times, and whole-cell lysates were separated by electrophoresis and immunoblotted with EBNA-2 antibody (upper panel). For the kinase assay, p34 cdc2 /cyclin B1-kinase complex was immunoprecipitated from the same cell lysates, and kinase activity was determined by [γ- 32 p]ATP incorporation into histone H1 (middle panel) and measured with a PhosphorImager (lower panel).

Techniques Used: Immunoprecipitation, Electrophoresis, Activity Assay, Kinase Assay

18) Product Images from "Casein Kinase II Phosphorylation Regulates αNAC Subcellular Localization and Transcriptional Coactivating Activity"

Article Title: Casein Kinase II Phosphorylation Regulates αNAC Subcellular Localization and Transcriptional Coactivating Activity

Journal: Gene Expression

doi:

Nuclear accumulation of αNAC after CK2 inhibition. (A) Quercetin or DRB treatment inhibits αNAC phosphorylation in cells. COS-7 cells transfected with pSI-NAC-Flag were treated for 2 h with the CK2 inhibitors, quercetin or DRB, or vehicle, and labeled with [γ- 32 P]ATP. Cellular extracts were immunoprecipitated with anti-Flag beads. Immunoprecipitates were analyzed by autoradiography and quantified by PhosphorImager analysis. Protein expression was normalized by Western blotting (not shown). *** p
Figure Legend Snippet: Nuclear accumulation of αNAC after CK2 inhibition. (A) Quercetin or DRB treatment inhibits αNAC phosphorylation in cells. COS-7 cells transfected with pSI-NAC-Flag were treated for 2 h with the CK2 inhibitors, quercetin or DRB, or vehicle, and labeled with [γ- 32 P]ATP. Cellular extracts were immunoprecipitated with anti-Flag beads. Immunoprecipitates were analyzed by autoradiography and quantified by PhosphorImager analysis. Protein expression was normalized by Western blotting (not shown). *** p

Techniques Used: Inhibition, Transfection, Labeling, Immunoprecipitation, Autoradiography, Expressing, Western Blot

αNAC is a phosphoprotein. (A) Amino acid sequence comparison of the αNAC proteins in vertebrate. αNAC from rat, chicken, and salmon are putative proteins. Amino acid residues 1–215 of mouse αNAC were aligned with αNAC proteins from human, rat, chicken, and salmon using the Clustal algorithm (MegAlign, DNASTAR Inc., Madison, WI). From human to salmon, a 99% to 83% identity was observed between the αNAC proteins. Nonconserved residues are shaded and conserved putative phosphoacceptor sites are boxed. Consensus sites for PKC, PKA, and CK2 are X-S/T-X-R/K, R-X1-2-S/T-X, and X-S/T-X-X-D/E, respectively. (B) αNAC is phosphorylated in COS-7 cells. COS-7 cells, transfected with pSI-NAC-Flag or pBlueScript (vector), were labeled with [γ- 32 P]ATP, and cellular extracts were immunoprecipitated with anti-Flag beads. Immunoprecipitates were analyzed by immunoblotting with the anti-NAC serum (lower panel) or by autoradiography (upper panel). M, molecular size markers in kDa.
Figure Legend Snippet: αNAC is a phosphoprotein. (A) Amino acid sequence comparison of the αNAC proteins in vertebrate. αNAC from rat, chicken, and salmon are putative proteins. Amino acid residues 1–215 of mouse αNAC were aligned with αNAC proteins from human, rat, chicken, and salmon using the Clustal algorithm (MegAlign, DNASTAR Inc., Madison, WI). From human to salmon, a 99% to 83% identity was observed between the αNAC proteins. Nonconserved residues are shaded and conserved putative phosphoacceptor sites are boxed. Consensus sites for PKC, PKA, and CK2 are X-S/T-X-R/K, R-X1-2-S/T-X, and X-S/T-X-X-D/E, respectively. (B) αNAC is phosphorylated in COS-7 cells. COS-7 cells, transfected with pSI-NAC-Flag or pBlueScript (vector), were labeled with [γ- 32 P]ATP, and cellular extracts were immunoprecipitated with anti-Flag beads. Immunoprecipitates were analyzed by immunoblotting with the anti-NAC serum (lower panel) or by autoradiography (upper panel). M, molecular size markers in kDa.

Techniques Used: Sequencing, Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, Autoradiography

19) Product Images from "Conformation-dependent phosphorylation of p53"

Article Title: Conformation-dependent phosphorylation of p53

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

( A ) Effect of WCE kinases on phosphorylation of p53. GST-p53 wild-type (amino acids 1–393; molecular mass 80 kDa) or truncated forms (as indicated by arrows) were incubated with WCE as source of kinases. The solid-phase kinase reaction initiated by addition of [γ- 32 P]ATP for 15 min at room temperature, followed by washes of glutathione beads-bound GST-p53 and elution of p53 from beads by SDS-loading buffer. Samples were separated on SDS/15% PAGE and autoradiographed. Molecular masses are indicated at left in kDa. GST-c-jun 5–89 used as control is shown in the left lane. ( B ) CKII phosphorylation of p53. Solid-phase kinase assay with respective GST-p53 proteins (position of major phosphorylation site indicated by arrow) was performed in the presence of 40 ng of the catalytic subunit of CKIIβ. ( C ) PKA phosphorylation of p53. GST-p53 proteins used for solid-phase kinase reaction using catalytic subunit of PKA (80 ng) as source of kinase. Position of major phosphorylation site is indicated by arrows. ( D ) Mapping of PKA phosphorylation sites on p53. Products of solid-phase kinase reaction using PKA (2 μg of enzyme per reaction) or WCE (2 μg) were washed and then separated on SDS/PAGE. Pattern of endogenous p53 phosphorylation is shown in IP lane, in which cells were labeled with [ 32 P]-orthophosphate (100 μCi) for 4 h before p53 was immunoprecipitated (200 ng of p53 antibody per 1 mg of protein). Bands corresponding to each of the GST-p53 proteins were excised and subjected to trypsinization followed by separation on an isoelectric focusing gel (pH range 3.5–10). Peptides were analyzed by autoradiography.
Figure Legend Snippet: ( A ) Effect of WCE kinases on phosphorylation of p53. GST-p53 wild-type (amino acids 1–393; molecular mass 80 kDa) or truncated forms (as indicated by arrows) were incubated with WCE as source of kinases. The solid-phase kinase reaction initiated by addition of [γ- 32 P]ATP for 15 min at room temperature, followed by washes of glutathione beads-bound GST-p53 and elution of p53 from beads by SDS-loading buffer. Samples were separated on SDS/15% PAGE and autoradiographed. Molecular masses are indicated at left in kDa. GST-c-jun 5–89 used as control is shown in the left lane. ( B ) CKII phosphorylation of p53. Solid-phase kinase assay with respective GST-p53 proteins (position of major phosphorylation site indicated by arrow) was performed in the presence of 40 ng of the catalytic subunit of CKIIβ. ( C ) PKA phosphorylation of p53. GST-p53 proteins used for solid-phase kinase reaction using catalytic subunit of PKA (80 ng) as source of kinase. Position of major phosphorylation site is indicated by arrows. ( D ) Mapping of PKA phosphorylation sites on p53. Products of solid-phase kinase reaction using PKA (2 μg of enzyme per reaction) or WCE (2 μg) were washed and then separated on SDS/PAGE. Pattern of endogenous p53 phosphorylation is shown in IP lane, in which cells were labeled with [ 32 P]-orthophosphate (100 μCi) for 4 h before p53 was immunoprecipitated (200 ng of p53 antibody per 1 mg of protein). Bands corresponding to each of the GST-p53 proteins were excised and subjected to trypsinization followed by separation on an isoelectric focusing gel (pH range 3.5–10). Peptides were analyzed by autoradiography.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Kinase Assay, SDS Page, Labeling, Immunoprecipitation, Autoradiography

( A ) Effect of DNA-binding domain-derived peptides on p53 phosphorylation. GST-p53 1–393 was subjected to solid-phase kinase reaction with WCE, in the presence of peptides 7–9 before addition of [γ 32 P]ATP (A lanes), before and during kinase reaction (B lanes), or only with the addition of [γ 32 P]ATP ( C ). Lower bands represent staining of the GST-p53 to indicate equal loading. ( B ) Effect of peptides on phosphorylation of p53 by PKA and WCE. GST-p53 1–393 was subjected to solid-phase phosphorylation (i.e., washes performed before addition of ATP; lanes C and 7–9) or with [γ 32 P]ATP added without intermediate washes (lanes A and 7*) by PKA or WCE, respectively, in the presence of indicated peptides. ( C ) Effect of P7 on p53 phosphorylation by CKIIβ, PKA, and WCE. GST-p53 subjected to solid-phase kinase reaction in the presence of P7 and kinases.
Figure Legend Snippet: ( A ) Effect of DNA-binding domain-derived peptides on p53 phosphorylation. GST-p53 1–393 was subjected to solid-phase kinase reaction with WCE, in the presence of peptides 7–9 before addition of [γ 32 P]ATP (A lanes), before and during kinase reaction (B lanes), or only with the addition of [γ 32 P]ATP ( C ). Lower bands represent staining of the GST-p53 to indicate equal loading. ( B ) Effect of peptides on phosphorylation of p53 by PKA and WCE. GST-p53 1–393 was subjected to solid-phase phosphorylation (i.e., washes performed before addition of ATP; lanes C and 7–9) or with [γ 32 P]ATP added without intermediate washes (lanes A and 7*) by PKA or WCE, respectively, in the presence of indicated peptides. ( C ) Effect of P7 on p53 phosphorylation by CKIIβ, PKA, and WCE. GST-p53 subjected to solid-phase kinase reaction in the presence of P7 and kinases.

Techniques Used: Binding Assay, Derivative Assay, Staining

20) Product Images from "The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide"

Article Title: The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Assay of endogenously formed ATP in the PSD by Ca 2+ /CaM-dependent phosphorylation of the PSD proteins. The experiments were performed as described. PSD fractions (100 μg each) were incubated at room temperature for 10 min with 32 Pi and ADP (lanes 1 and 2) and the mixtures were processed for phosphorylation in the absence (−) or presence (+) of Ca 2+ /CaM. Control experiments were examined in parallel in which the Ca 2+ /CaM-dependent phosphorylation of the PSD samples was performed by using three doses of exogenously added [γ- 32 P]ATP (lanes 3 and 4, 8.3 fmol each; lanes 5 and 6, 16.6 fmol each; lanes 7 and 8, 33.20 fmol each). The mixtures were then subjected to SDS/PAGE for autoradiographic analysis. mPSDp is the major PSD protein.
Figure Legend Snippet: Assay of endogenously formed ATP in the PSD by Ca 2+ /CaM-dependent phosphorylation of the PSD proteins. The experiments were performed as described. PSD fractions (100 μg each) were incubated at room temperature for 10 min with 32 Pi and ADP (lanes 1 and 2) and the mixtures were processed for phosphorylation in the absence (−) or presence (+) of Ca 2+ /CaM. Control experiments were examined in parallel in which the Ca 2+ /CaM-dependent phosphorylation of the PSD samples was performed by using three doses of exogenously added [γ- 32 P]ATP (lanes 3 and 4, 8.3 fmol each; lanes 5 and 6, 16.6 fmol each; lanes 7 and 8, 33.20 fmol each). The mixtures were then subjected to SDS/PAGE for autoradiographic analysis. mPSDp is the major PSD protein.

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

21) Product Images from "The U69 Gene of Human Herpesvirus 6 Encodes a Protein Kinase Which Can Confer Ganciclovir Sensitivity to Baculoviruses"

Article Title: The U69 Gene of Human Herpesvirus 6 Encodes a Protein Kinase Which Can Confer Ganciclovir Sensitivity to Baculoviruses

Journal: Journal of Virology

doi:

Phosphoamino acid analysis of recombinant U69. U69 was partially purified with Ni 2+ -NTA and phosphorylated in vitro in the presence of [γ- 32 P]ATP. The proteins were separated by SDS-PAGE and transferred electrophoretically onto a PVDF membrane, and the band corresponding to phosphorylated U69 was excised and acid hydrolyzed. The hydrolysate was mixed with unlabelled phosphoamino acids, phosphoserine (P-SER), phosphothreonine (P-THR), and phosphotyrosine (P-TYR) and subjected to two-dimensional electrophoretic thin-layer chromatography. The unlabelled phosphoamino acids were visualized with ninhydrin, and the autoradiograph of the thin-layer chromatography plate for the phosphoaminoacid analysis of HHV-6A U69 is shown. Phosphoamino acid analysis of HHV-6B U69 revealed a similar autoradiograph. The labelled phosphoamino acids comigrated with P-SER and P-THR, and the migration of P-TYR is indicated by the dotted circle.
Figure Legend Snippet: Phosphoamino acid analysis of recombinant U69. U69 was partially purified with Ni 2+ -NTA and phosphorylated in vitro in the presence of [γ- 32 P]ATP. The proteins were separated by SDS-PAGE and transferred electrophoretically onto a PVDF membrane, and the band corresponding to phosphorylated U69 was excised and acid hydrolyzed. The hydrolysate was mixed with unlabelled phosphoamino acids, phosphoserine (P-SER), phosphothreonine (P-THR), and phosphotyrosine (P-TYR) and subjected to two-dimensional electrophoretic thin-layer chromatography. The unlabelled phosphoamino acids were visualized with ninhydrin, and the autoradiograph of the thin-layer chromatography plate for the phosphoaminoacid analysis of HHV-6A U69 is shown. Phosphoamino acid analysis of HHV-6B U69 revealed a similar autoradiograph. The labelled phosphoamino acids comigrated with P-SER and P-THR, and the migration of P-TYR is indicated by the dotted circle.

Techniques Used: Phosphoamino Acid Analysis, Recombinant, Purification, In Vitro, SDS Page, Thin Layer Chromatography, Autoradiography, Migration

Phosphorylation of purified recombinant U69. Equal amounts of U69, purified from BV6AU69- or BV6BU69-infected insect cells, were incubated with [γ- 32 P]ATP in a protein kinase assay and subjected to SDS-PAGE (lanes 2 and 3, respectively). The gel was dried and exposed to film. The resulting autoradiograph is shown. Lane 4 is immobilized U69-specific IgG used for immunoprecipitation of BVLacZ-infected insect cells and subjected to a protein kinase assay as described above. The molecular sizes of the protein markers (lane M) are indicated on the left.
Figure Legend Snippet: Phosphorylation of purified recombinant U69. Equal amounts of U69, purified from BV6AU69- or BV6BU69-infected insect cells, were incubated with [γ- 32 P]ATP in a protein kinase assay and subjected to SDS-PAGE (lanes 2 and 3, respectively). The gel was dried and exposed to film. The resulting autoradiograph is shown. Lane 4 is immobilized U69-specific IgG used for immunoprecipitation of BVLacZ-infected insect cells and subjected to a protein kinase assay as described above. The molecular sizes of the protein markers (lane M) are indicated on the left.

Techniques Used: Purification, Recombinant, Infection, Incubation, Protein Kinase Assay, SDS Page, Autoradiography, Immunoprecipitation

22) Product Images from "pTAR-Encoded Proteins in Plasmid Partitioning"

Article Title: pTAR-Encoded Proteins in Plasmid Partitioning

Journal: Journal of Bacteriology

doi:

Stimulation of the ATPase activity of ParA by plasmid DNA. ATPase activity was measured as the release of 32 PO 4 from [γ- 32 P]ATP by thin-layer chromatography as described in Materials and Methods. Shown are results with pUCD550 (containing parS ). Identical results (data not shown) were obtained with the vector pUC4.
Figure Legend Snippet: Stimulation of the ATPase activity of ParA by plasmid DNA. ATPase activity was measured as the release of 32 PO 4 from [γ- 32 P]ATP by thin-layer chromatography as described in Materials and Methods. Shown are results with pUCD550 (containing parS ). Identical results (data not shown) were obtained with the vector pUC4.

Techniques Used: Activity Assay, Plasmid Preparation, Thin Layer Chromatography

23) Product Images from "Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes"

Article Title: Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Journal: Molecular Biology of the Cell

doi:

Kinase activities in GPI-enriched membranes isolated using different detergents. Buoyant, GPI-enriched membranes (0.5 ml, equivalent to ∼25 × 10 6 cells) from the same cell homogenate, untreated (None) or after treatment with indicated detergents (1% TX-100, 60 mM OTG, 0.5% NP-40, or 20 mM CHAPS), were diluted in TKM buffer, and the GPI-enriched membranes sedimented at 250,000 × g for 4 h were suspended in kinase buffer. (A) Total protein profile after silver staining. (B) Western blot detection of Thy-1, Lck, and Fyn in 20 μl of the GPI-enriched membranes shown in A. (C) Phosphoprotein profile after in vitro phosphorylation in the presence of [γ- 32 P]ATP as described in MATERIALS AND METHODS. Phosphorylated proteins were separated in 5–20% SDS-PAGE gradient gels and detected by autoradiography after 4.5 h of exposure. For comparison a 20-h exposure of the first two lanes (none and TX-100) is shown in the right panel. Data representative of three independent experiments are shown. (D) Western blot detection of tyrosine-phosphorylated proteins with 4G10 anti-phosphotyrosine mAb after in vitro kinase assay on untreated and TX-100–treated GPI-enriched membranes in the absence (−) or presence (+) of 50 μM cold ATP. The hyperphosphorylated kinase bands are marked by arrowheads.
Figure Legend Snippet: Kinase activities in GPI-enriched membranes isolated using different detergents. Buoyant, GPI-enriched membranes (0.5 ml, equivalent to ∼25 × 10 6 cells) from the same cell homogenate, untreated (None) or after treatment with indicated detergents (1% TX-100, 60 mM OTG, 0.5% NP-40, or 20 mM CHAPS), were diluted in TKM buffer, and the GPI-enriched membranes sedimented at 250,000 × g for 4 h were suspended in kinase buffer. (A) Total protein profile after silver staining. (B) Western blot detection of Thy-1, Lck, and Fyn in 20 μl of the GPI-enriched membranes shown in A. (C) Phosphoprotein profile after in vitro phosphorylation in the presence of [γ- 32 P]ATP as described in MATERIALS AND METHODS. Phosphorylated proteins were separated in 5–20% SDS-PAGE gradient gels and detected by autoradiography after 4.5 h of exposure. For comparison a 20-h exposure of the first two lanes (none and TX-100) is shown in the right panel. Data representative of three independent experiments are shown. (D) Western blot detection of tyrosine-phosphorylated proteins with 4G10 anti-phosphotyrosine mAb after in vitro kinase assay on untreated and TX-100–treated GPI-enriched membranes in the absence (−) or presence (+) of 50 μM cold ATP. The hyperphosphorylated kinase bands are marked by arrowheads.

Techniques Used: Isolation, Silver Staining, Western Blot, In Vitro, SDS Page, Autoradiography, Kinase Assay

24) Product Images from "Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase"

Article Title: Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Journal: Molecular Biology of the Cell

doi:

Protein kinase activity of ANPK. CHO cells were transiently transfected with 10 μg of expression vectors for FLAG-tagged ANPK(159–1191) and ANPK(K226R), in which Lys 226 in the ATP-binding site was converted to an Arg. Proteins were immunoprecipitated using anti-FLAG mAb and subjected to immune complex kinase assays. (A) Phosphorylation of myelin basic protein (MBP, 5 μM) by immunopurified ANPK(159–1191) (lane 1), but not by ANPK(K226R) (lane 3), ANPK(159–1191), and ANPK(K226R) in the absence of MBP (lanes 2 and 4, respectively). (B) Autophosphorylation by ANPK(159–1191) (lanes 2 and 3) and ANPK(K226R) (lanes 1 and 4). (C) Protein kinase activity of purified GST-ANPK(159–920) expressed in E. coli. GST-ANPK(159–920) with MBP (lane 1), autophosphorylation of GST-ANPK(159–920) in the absence of MBP (lane 3), and MBP in the absence of GST-ANPK(159–920) (lane 2). (D) Comparison of MBP (lane 1), c-Jun (lane 2), histone H3 (lane 3), histone H1 (lane 4), and GST-ANPK(802–920) (lane 5) as substrates for GST-ANPK(159–920). Reaction mixture (20 μl) contained 0.7 μg of GST-ANPK(159–920) and 5 μM substrate. After 30°C for 30 min, the reactions were terminated by addition of 20 μl of 2× Laemmli sample buffer, after which the products were subjected to electrophoresis under denaturing conditions on 15% (A, C, and D) or 7.5% polyacrylamide gels (B) and visualized by autoradiography. (F) Solid-phase phosphorylation of synthetic peptides by ANPK. PhosphoSpots test strip (Jerini Bio Tools) containing covalently bound substrate peptides for indicated kinases was phosphorylated by incubating with 4 μg of GST-ANPK(159–920) in 500 μl of 2× kinase buffer containing 100 μM [γ- 32 P]ATP at 22°C for 20 min. The reaction was stopped and filter washed extensively according to manufacturer’s instructions and analyzed by autoradiography. (The peptide no. 19 contains both Ser and Tyr residues as potential phosphoacceptor sites.)
Figure Legend Snippet: Protein kinase activity of ANPK. CHO cells were transiently transfected with 10 μg of expression vectors for FLAG-tagged ANPK(159–1191) and ANPK(K226R), in which Lys 226 in the ATP-binding site was converted to an Arg. Proteins were immunoprecipitated using anti-FLAG mAb and subjected to immune complex kinase assays. (A) Phosphorylation of myelin basic protein (MBP, 5 μM) by immunopurified ANPK(159–1191) (lane 1), but not by ANPK(K226R) (lane 3), ANPK(159–1191), and ANPK(K226R) in the absence of MBP (lanes 2 and 4, respectively). (B) Autophosphorylation by ANPK(159–1191) (lanes 2 and 3) and ANPK(K226R) (lanes 1 and 4). (C) Protein kinase activity of purified GST-ANPK(159–920) expressed in E. coli. GST-ANPK(159–920) with MBP (lane 1), autophosphorylation of GST-ANPK(159–920) in the absence of MBP (lane 3), and MBP in the absence of GST-ANPK(159–920) (lane 2). (D) Comparison of MBP (lane 1), c-Jun (lane 2), histone H3 (lane 3), histone H1 (lane 4), and GST-ANPK(802–920) (lane 5) as substrates for GST-ANPK(159–920). Reaction mixture (20 μl) contained 0.7 μg of GST-ANPK(159–920) and 5 μM substrate. After 30°C for 30 min, the reactions were terminated by addition of 20 μl of 2× Laemmli sample buffer, after which the products were subjected to electrophoresis under denaturing conditions on 15% (A, C, and D) or 7.5% polyacrylamide gels (B) and visualized by autoradiography. (F) Solid-phase phosphorylation of synthetic peptides by ANPK. PhosphoSpots test strip (Jerini Bio Tools) containing covalently bound substrate peptides for indicated kinases was phosphorylated by incubating with 4 μg of GST-ANPK(159–920) in 500 μl of 2× kinase buffer containing 100 μM [γ- 32 P]ATP at 22°C for 20 min. The reaction was stopped and filter washed extensively according to manufacturer’s instructions and analyzed by autoradiography. (The peptide no. 19 contains both Ser and Tyr residues as potential phosphoacceptor sites.)

Techniques Used: Activity Assay, Transfection, Expressing, Binding Assay, Immunoprecipitation, Immune Complex Kinase Assay, Purification, Electrophoresis, Autoradiography, Stripping Membranes

25) Product Images from "E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs"

Article Title: E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs

Journal: Journal of Virology

doi:

E1B-AP5 cDNA maps, nucleotide sequence, and predicted amino acid sequence. (A) cDNA clones E1B-AP5/1 and E1B-AP5/2 were isolated with the [γ- 32 P]ATP-labeled GSTE1B-55kDa protein probe from a λgt11 HeLa cDNA expression library. cDNA clones E1B-AP5/3 to E1B-AP5/7 were isolated from the same library by rescreening with E1B-AP5/2 and later with a fragment from the 3′ end of E1B-AP5/3. The thick black bar on top represents the 2,568-bp open reading frame of E1B-AP5. Thin bars denote the 5′ and 3′ untranslated sequences, respectively. The locations of some unique restriction enzymes are indicated above the bars. (B) The complete E1B-AP5 cDNA sequence was generated by assembling restriction fragments from E1B-AP5/6 and E1B-AP5/7 in pBKRSV. Sequence was determined from each cDNA clone twice on both strands by sequence-derived oligonucleotide primers. The predicted amino acid sequence is shown in the single-letter code.
Figure Legend Snippet: E1B-AP5 cDNA maps, nucleotide sequence, and predicted amino acid sequence. (A) cDNA clones E1B-AP5/1 and E1B-AP5/2 were isolated with the [γ- 32 P]ATP-labeled GSTE1B-55kDa protein probe from a λgt11 HeLa cDNA expression library. cDNA clones E1B-AP5/3 to E1B-AP5/7 were isolated from the same library by rescreening with E1B-AP5/2 and later with a fragment from the 3′ end of E1B-AP5/3. The thick black bar on top represents the 2,568-bp open reading frame of E1B-AP5. Thin bars denote the 5′ and 3′ untranslated sequences, respectively. The locations of some unique restriction enzymes are indicated above the bars. (B) The complete E1B-AP5 cDNA sequence was generated by assembling restriction fragments from E1B-AP5/6 and E1B-AP5/7 in pBKRSV. Sequence was determined from each cDNA clone twice on both strands by sequence-derived oligonucleotide primers. The predicted amino acid sequence is shown in the single-letter code.

Techniques Used: Sequencing, Clone Assay, Isolation, Labeling, Expressing, Generated, Derivative Assay

26) Product Images from "Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts"

Article Title: Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

Journal: Molecular and Cellular Biology

doi:

TLS past and AAF adduct by extracts from normal or XPV cells. Analysis of TLS catalyzed by 30 μg of either normal (N; 205BR) or XPV (XP6DU) cell extracts. DNA products obtained after 1 h of incubation with pUC-3G1.ss or pUC-3G3.ss were cleaved with enzymes Pvu II and Eco RI and subjected to electrophoresis on an 10% polyacrylamide–7 M urea denaturing gel. L−1, L0, and L+1 are products generated if synthesis is blocked one nucleotide before, opposite, and one nucleotide after the lesion, respectively. TLS0 and TLS−1 are products from TLS via nonslipped and slipped intermediates. As an internal standard, an identical amount of a 120-nucleotide fragment (end labeled with [γ- 32 P]ATP by T4 polynucleotide kinase) was added to each reaction at the end of the replication step.
Figure Legend Snippet: TLS past and AAF adduct by extracts from normal or XPV cells. Analysis of TLS catalyzed by 30 μg of either normal (N; 205BR) or XPV (XP6DU) cell extracts. DNA products obtained after 1 h of incubation with pUC-3G1.ss or pUC-3G3.ss were cleaved with enzymes Pvu II and Eco RI and subjected to electrophoresis on an 10% polyacrylamide–7 M urea denaturing gel. L−1, L0, and L+1 are products generated if synthesis is blocked one nucleotide before, opposite, and one nucleotide after the lesion, respectively. TLS0 and TLS−1 are products from TLS via nonslipped and slipped intermediates. As an internal standard, an identical amount of a 120-nucleotide fragment (end labeled with [γ- 32 P]ATP by T4 polynucleotide kinase) was added to each reaction at the end of the replication step.

Techniques Used: Incubation, Electrophoresis, Generated, Labeling

27) Product Images from "Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt"

Article Title: Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Journal: Molecular and Cellular Biology

doi:

Stimulation of fTie2 in vivo induces phosphorylation and activation of Akt. (A) Phosphorylation and activation of Akt requires an intact Y1101 residue on Tie2. Untransfected NIH 3T3 cells or those expressing fTie2 chimeric receptors were left unstimulated or stimulated with CSF-1 (500 ng/ml), all in the presence of 1 mM sodium orthovanadate. Aliquots of total cell lysates were separated by SDS–8% PAGE and blotted with anti-Tie2 PAb to demonstrate similar receptor expression levels (top) and anti-Akt PAb to evaluate Akt expression and demonstrate gel mobility shift secondary to phosphorylation (middle). Anti-Akt immunoprecipitates from the same cell lysates were used in kinase assays with the substrate histone H2B and [γ- 32 P]ATP. Proteins were separated by SDS–15% PAGE and evaluated by autoradiography to assess Akt activation after CSF-1 stimulation (bottom). (B) Tie2-mediated phosphorylation and activation of Akt is PI3-kinase dependent. Fibroblasts expressing wild-type fTie2 or Y1112F were either unstimulated, stimulated with CSF-1, or stimulated with CSF-1 following 15 min of pretreatment with 100 nM wortmannin, all in the presence of 1 mM vanadate. Untransfected 3T3 cells were treated similarly with PDGF-BB (30 ng/ml) as a positive control. Cell lysates were used to assess Akt phosphorylation by Western blotting and in kinase assays to evaluate Akt activation as described for panel A. (C) Wild-type- and mutant fTie2-expressing cells exhibit intact Akt signaling pathways. Untransfected 3T3 cells and cells expressing wild-type or mutant fTie2 receptors were evaluated as described for panel A, except that stimulation of all cells was with PDGF (30 ng/ml) to demonstrate intact signaling pathways upstream of Akt. The positions of Akt, phosphorylated Akt (pAkt), and histone H2B are shown.
Figure Legend Snippet: Stimulation of fTie2 in vivo induces phosphorylation and activation of Akt. (A) Phosphorylation and activation of Akt requires an intact Y1101 residue on Tie2. Untransfected NIH 3T3 cells or those expressing fTie2 chimeric receptors were left unstimulated or stimulated with CSF-1 (500 ng/ml), all in the presence of 1 mM sodium orthovanadate. Aliquots of total cell lysates were separated by SDS–8% PAGE and blotted with anti-Tie2 PAb to demonstrate similar receptor expression levels (top) and anti-Akt PAb to evaluate Akt expression and demonstrate gel mobility shift secondary to phosphorylation (middle). Anti-Akt immunoprecipitates from the same cell lysates were used in kinase assays with the substrate histone H2B and [γ- 32 P]ATP. Proteins were separated by SDS–15% PAGE and evaluated by autoradiography to assess Akt activation after CSF-1 stimulation (bottom). (B) Tie2-mediated phosphorylation and activation of Akt is PI3-kinase dependent. Fibroblasts expressing wild-type fTie2 or Y1112F were either unstimulated, stimulated with CSF-1, or stimulated with CSF-1 following 15 min of pretreatment with 100 nM wortmannin, all in the presence of 1 mM vanadate. Untransfected 3T3 cells were treated similarly with PDGF-BB (30 ng/ml) as a positive control. Cell lysates were used to assess Akt phosphorylation by Western blotting and in kinase assays to evaluate Akt activation as described for panel A. (C) Wild-type- and mutant fTie2-expressing cells exhibit intact Akt signaling pathways. Untransfected 3T3 cells and cells expressing wild-type or mutant fTie2 receptors were evaluated as described for panel A, except that stimulation of all cells was with PDGF (30 ng/ml) to demonstrate intact signaling pathways upstream of Akt. The positions of Akt, phosphorylated Akt (pAkt), and histone H2B are shown.

Techniques Used: In Vivo, Activation Assay, Expressing, Polyacrylamide Gel Electrophoresis, Mobility Shift, Autoradiography, Positive Control, Western Blot, Mutagenesis

28) Product Images from "Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1?"

Article Title: Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1?

Journal: American journal of physiology. Gastrointestinal and liver physiology

doi: 10.1152/ajpgi.00121.2004

EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.
Figure Legend Snippet: EMSA analysis of HNF-1 binding to the distal HNF-1 response element in conserved cluster I of the calbindin D 9k promoter. Oligonucleotides containing the putative HNF-1 site from cluster I in the human calbindin D 9k promoter (CaBP D 9k ) or a well-characterized HNF-1 site from the lactase promoter (Lactase) were labeled with [γ- 32 P]ATP by T4 polynucleotide kinase. Left : competitive gel-shift assay. Nuclear extracts (Nuc Extract; 10 µg) from preconfluent (2 days) or 5-day postconfluent (9 days) TC7 cells were used for each binding reaction. The specific HNF-1α-containing complex is marked with an arrow. Specificity of complex formation was confirmed by competition with a 6-fold (6×) or 30-fold (30×) molar excess of unlabeled probe (9k, CaBP-HNF-1; M, mutated CaBP-HNF-1; L, lactase HNF-1; A, AP2). Specificity of complex formation on the labeled lactase HNF-1 probe was confirmed by competition with a 50-fold molar excess of unlabeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe. Right , HNF-1α supershift assay. Nuclear extracts (10 µg) from 9-day cultures of TC7 cells were preincubated with HNF-1α antibody (H) or a goat IgG (Ig) before incubation with 32 P-labeled calbindin D 9k HNF-1 probe or lactase HNF-1 probe.

Techniques Used: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Incubation

29) Product Images from "Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator"

Article Title: Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator

Journal: The EMBO Journal

doi: 10.1093/emboj/19.5.1068

Fig. 8. Gel retardation analysis with purified CrgA. The promoter regions used were those of crgA , pilC1 and pilT . These regions were amplified by PCR using oligonucleotides 98-2/98-3 and 98-1/98-3 ( crgA ), C1-354/C1-8 and C1-128/C1-8 ( pilC1 ) and 99-17 and 99-18 ( pilT ). PCR fragments were end labelled using T4 polynucleotide kinase and [γ- 32 P]ATP (3000 Ci/mmol; Amersham). For each sample, 25 ng of labelled fragment were incubated with increasing amounts of purified CrgA (top) as described in Materials and methods. Heterologous DNA (500 ng) was present in all cases. Competition experiments with unlabelled homologous DNA (100 ng) were also performed for crgA and pilC1 .
Figure Legend Snippet: Fig. 8. Gel retardation analysis with purified CrgA. The promoter regions used were those of crgA , pilC1 and pilT . These regions were amplified by PCR using oligonucleotides 98-2/98-3 and 98-1/98-3 ( crgA ), C1-354/C1-8 and C1-128/C1-8 ( pilC1 ) and 99-17 and 99-18 ( pilT ). PCR fragments were end labelled using T4 polynucleotide kinase and [γ- 32 P]ATP (3000 Ci/mmol; Amersham). For each sample, 25 ng of labelled fragment were incubated with increasing amounts of purified CrgA (top) as described in Materials and methods. Heterologous DNA (500 ng) was present in all cases. Competition experiments with unlabelled homologous DNA (100 ng) were also performed for crgA and pilC1 .

Techniques Used: Electrophoretic Mobility Shift Assay, Purification, Amplification, Polymerase Chain Reaction, Incubation

30) Product Images from "A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver"

Article Title: A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.161284298

( A ) ChREBP contains several recognizable protein motifs, including a bipartite nuclear localization signal (NLS), a basic helix–loop–helix leucine zipper (bHLH-ZIP) motif, and three consensus PKA phosphorylation sites (P) including one (sequence RRIT) within the bHLH region. ( B ) ( Left ) DNA-binding activity of ChREBP can be regulated by phosphorylation in vitro . Incubation of active ChREBP (lane 1) for 20 min with both ATP (1 mM) and PKA (0.1 unit/μl) abolished DNA-binding activity (lane 2) as measured by gel-shift analysis with the 32 P-radiolabeled WT oligonucleotide. Omission of ATP from the reaction mixture prevents ChREBP inactivation (lane 3). PKA-dependent ChREBP inactivation is blocked by 0.2 unit/μl protein kinase inhibitor (PKI) (lane 4). After PKI addition, DNA-binding activity of phosphorylated ChREBP can be restored by addition of 0.025 unit/μl protein phosphatase 2A (PP2A) (lane 5). Addition of the PP2A inhibitor okadaic acid (OKA) (10 nM) prevents reactivation of the ChREBP DNA-binding activity. ( Right ) Incubation of the protein with [γ- 32 P]ATP in the presence of PKA leads to radiolabeling of ChREBP observed after SDS/PAGE.
Figure Legend Snippet: ( A ) ChREBP contains several recognizable protein motifs, including a bipartite nuclear localization signal (NLS), a basic helix–loop–helix leucine zipper (bHLH-ZIP) motif, and three consensus PKA phosphorylation sites (P) including one (sequence RRIT) within the bHLH region. ( B ) ( Left ) DNA-binding activity of ChREBP can be regulated by phosphorylation in vitro . Incubation of active ChREBP (lane 1) for 20 min with both ATP (1 mM) and PKA (0.1 unit/μl) abolished DNA-binding activity (lane 2) as measured by gel-shift analysis with the 32 P-radiolabeled WT oligonucleotide. Omission of ATP from the reaction mixture prevents ChREBP inactivation (lane 3). PKA-dependent ChREBP inactivation is blocked by 0.2 unit/μl protein kinase inhibitor (PKI) (lane 4). After PKI addition, DNA-binding activity of phosphorylated ChREBP can be restored by addition of 0.025 unit/μl protein phosphatase 2A (PP2A) (lane 5). Addition of the PP2A inhibitor okadaic acid (OKA) (10 nM) prevents reactivation of the ChREBP DNA-binding activity. ( Right ) Incubation of the protein with [γ- 32 P]ATP in the presence of PKA leads to radiolabeling of ChREBP observed after SDS/PAGE.

Techniques Used: Sequencing, Binding Assay, Activity Assay, In Vitro, Incubation, Electrophoretic Mobility Shift Assay, Radioactivity, SDS Page

31) Product Images from "Modulation of telomere protection by the PI3K/AKT pathway"

Article Title: Modulation of telomere protection by the PI3K/AKT pathway

Journal: Nature Communications

doi: 10.1038/s41467-017-01329-2

TRF1 is phosphorylated in vitro by AKT1. a One μg of purified GST-TRF1 was incubated with 0.2 μg of human GST-AKT1 with or without AKT inhibitor (MK-2206) in kinase buffer containing 5 μCi [γ- 32 -P]ATP (left). The peptide RBER-GSK3(14-27) was used as positive controls (right). The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. b MS/MS spectrum of unmodified TRF1 328-339 (above panel) and of the TRF1 328-339 peptide in which phosphorylation was assigned to T330 residue (below panel). c MS/MS spectrum of unmodified TRF1 246-255 peptide and of the TRF1 246-255 peptide in which phosphorylation was unambiguously assigned to the T248 residue (below panel). Mass error, identification score, and posterior error probability (PEP) are shown for the different peptides. d , e Quantification of TGTLQCETTMER (T330) ( d ) and of the AATKVVENEK (T248) ( e ). Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus AKT1. f Schematic representation of TRF1 protein depicting its N-terminal acidic domain, TRFH domain, nuclear localization signal (NLS), and its Myb domain. The residues found phosphorylated by AKT (T248, T330, and S344) were mutated to alanine. g Representative image of in vitro phosphorylation assay of purified GST-TRF1, GST-TRF1(T330A), GST-TRF1 (S344A), GST-TRF1 (T248A), or GST-TRF1 (T330A/S344A/T248A) by GST-AKT1. The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. As loading control, purified GST-TRF1 protein samples were run in SDS-PAGE and coomassie-stained (below panel). The quantification of mutant P-TRF1 levels normalized to wild-type TRF1 is shown to the right. Student’s t test was used for statistical analysis, P values are shown. Error bars represent standard deviation. n number of independent experiments
Figure Legend Snippet: TRF1 is phosphorylated in vitro by AKT1. a One μg of purified GST-TRF1 was incubated with 0.2 μg of human GST-AKT1 with or without AKT inhibitor (MK-2206) in kinase buffer containing 5 μCi [γ- 32 -P]ATP (left). The peptide RBER-GSK3(14-27) was used as positive controls (right). The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. b MS/MS spectrum of unmodified TRF1 328-339 (above panel) and of the TRF1 328-339 peptide in which phosphorylation was assigned to T330 residue (below panel). c MS/MS spectrum of unmodified TRF1 246-255 peptide and of the TRF1 246-255 peptide in which phosphorylation was unambiguously assigned to the T248 residue (below panel). Mass error, identification score, and posterior error probability (PEP) are shown for the different peptides. d , e Quantification of TGTLQCETTMER (T330) ( d ) and of the AATKVVENEK (T248) ( e ). Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus AKT1. f Schematic representation of TRF1 protein depicting its N-terminal acidic domain, TRFH domain, nuclear localization signal (NLS), and its Myb domain. The residues found phosphorylated by AKT (T248, T330, and S344) were mutated to alanine. g Representative image of in vitro phosphorylation assay of purified GST-TRF1, GST-TRF1(T330A), GST-TRF1 (S344A), GST-TRF1 (T248A), or GST-TRF1 (T330A/S344A/T248A) by GST-AKT1. The reaction mixtures were resolved by SDS-PAGE and subjected to autoradiography. As loading control, purified GST-TRF1 protein samples were run in SDS-PAGE and coomassie-stained (below panel). The quantification of mutant P-TRF1 levels normalized to wild-type TRF1 is shown to the right. Student’s t test was used for statistical analysis, P values are shown. Error bars represent standard deviation. n number of independent experiments

Techniques Used: In Vitro, Purification, Incubation, SDS Page, Autoradiography, Mass Spectrometry, Phosphorylation Assay, Staining, Mutagenesis, Standard Deviation

32) Product Images from "Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains"

Article Title: Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains

Journal: Journal of Virology

doi: 10.1128/JVI.76.7.3461-3470.2002

NSP5 is a substrate of CKII. Shown is SDS-PAGE analysis of an in vitro CKII phosphorylation assay with [γ- 32 P]ATP. (A) The indicated GST-NSP5 mutants were used as substrates. Positive and negative controls are shown in lanes 1, 7, 8, and 9. −, no substrate added. (B) Purified, in vitro-translated 35 S-His 6 -Δ1 was used as a substrate in the presence or absence of CKII. The two panels show autoradiography of the same gel, detecting 35 S plus 32 P (left) and 32 P (right). The solid and open arrowheads indicate His 6 -Δ1 precursor and hyperphosphorylated forms, respectively. Autophosphorylated GST-α and GST-β CKII subunits are indicated. +, present; −, absent.
Figure Legend Snippet: NSP5 is a substrate of CKII. Shown is SDS-PAGE analysis of an in vitro CKII phosphorylation assay with [γ- 32 P]ATP. (A) The indicated GST-NSP5 mutants were used as substrates. Positive and negative controls are shown in lanes 1, 7, 8, and 9. −, no substrate added. (B) Purified, in vitro-translated 35 S-His 6 -Δ1 was used as a substrate in the presence or absence of CKII. The two panels show autoradiography of the same gel, detecting 35 S plus 32 P (left) and 32 P (right). The solid and open arrowheads indicate His 6 -Δ1 precursor and hyperphosphorylated forms, respectively. Autophosphorylated GST-α and GST-β CKII subunits are indicated. +, present; −, absent.

Techniques Used: SDS Page, In Vitro, Phosphorylation Assay, Purification, Autoradiography

33) Product Images from "Reciprocal regulation of expression of the human adenosine 5?-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors"

Article Title: Reciprocal regulation of expression of the human adenosine 5?-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors

Journal: Nucleic Acids Research

doi:

S1 nuclease protection mapping of the ABCA2 transcription start site. A 5′-end phosphorylated 479 bp double-stranded DNA template for the S1 probe was generated by PCR and digested with λ exonuclease to generate an antisense single-stranded DNA probe that was end labeled with [γ- 32 P]ATP and hybridized to total RNA from BE(2)-M17 neuroblastoma cells. Following digestion with S1 nuclease, the protected fragment was separated by electrophoresis in a 6% polyacrylamide–7 M urea gel followed by autoradiography. The antisense strand of the 479 bp DNA template was manually sequenced and the φX174 Hin f DNA ladder was radiolabeled with [γ- 32 P]ATP to serve as sequence and size markers respectively. An arrow indicates a major protected fragment of 152 bp, located 95 bp upstream of the ATG translation start site that represents the major transcription start site.
Figure Legend Snippet: S1 nuclease protection mapping of the ABCA2 transcription start site. A 5′-end phosphorylated 479 bp double-stranded DNA template for the S1 probe was generated by PCR and digested with λ exonuclease to generate an antisense single-stranded DNA probe that was end labeled with [γ- 32 P]ATP and hybridized to total RNA from BE(2)-M17 neuroblastoma cells. Following digestion with S1 nuclease, the protected fragment was separated by electrophoresis in a 6% polyacrylamide–7 M urea gel followed by autoradiography. The antisense strand of the 479 bp DNA template was manually sequenced and the φX174 Hin f DNA ladder was radiolabeled with [γ- 32 P]ATP to serve as sequence and size markers respectively. An arrow indicates a major protected fragment of 152 bp, located 95 bp upstream of the ATG translation start site that represents the major transcription start site.

Techniques Used: Generated, Polymerase Chain Reaction, Labeling, Electrophoresis, Autoradiography, Sequencing

34) Product Images from "Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase"

Article Title: Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Journal: Purinergic Signalling

doi: 10.1007/s11302-010-9203-x

Ebselen inhibits the UDP-dependent conversion of [ γ 32 P]ATP to [ 32 P]UTP on WD-HBE cells. HPLC analysis of [ γ 32 P]ATP metabolism on WD-HBE cells. Radiotracer [ γ 32 P]ATP (∼0.1 μCi) was added to cultures (mucosal addition) and the resulting [ 32 ”. a Representative HPLC tracings ( n = 4) corresponding to samples incubated for T = 0 min ( i ), or 3 min in the presence of vehicle ( ii ), 30 μM ebselen ( Ebs ) ( iii ), 10 μM UDP ( iv ), or 30 μM ebselen and 10 μM UDP ( v ). The tracings represent the radioactivity ( cpm ) eluting from the HPLC column, recorded at 10-s intervals. b [ γ 32 P]ATP alone ( i ), [ γ 32 P]ATP and 10 μM UDP ( ii ), or [ γ 32 P]ATP and 10 μM AMP ( iii ) was added to cell cultures or to conditioned medium (Con. medium) for the indicated times, and the formation of 32 Pi (i), [ 32 P]UTP ( ii ), and [ 32 P]ADP ( iii ) were quantified by HPLC (mean ± SD from two experiments performed in triplicates). The results are expressed as percent conversion of [ 32 P]ATP to [ 32 P]-product, relative to T = 0”. The area of each radioactive peak was calculated using the HPLC analysis software provided by the manufacturer
Figure Legend Snippet: Ebselen inhibits the UDP-dependent conversion of [ γ 32 P]ATP to [ 32 P]UTP on WD-HBE cells. HPLC analysis of [ γ 32 P]ATP metabolism on WD-HBE cells. Radiotracer [ γ 32 P]ATP (∼0.1 μCi) was added to cultures (mucosal addition) and the resulting [ 32 ”. a Representative HPLC tracings ( n = 4) corresponding to samples incubated for T = 0 min ( i ), or 3 min in the presence of vehicle ( ii ), 30 μM ebselen ( Ebs ) ( iii ), 10 μM UDP ( iv ), or 30 μM ebselen and 10 μM UDP ( v ). The tracings represent the radioactivity ( cpm ) eluting from the HPLC column, recorded at 10-s intervals. b [ γ 32 P]ATP alone ( i ), [ γ 32 P]ATP and 10 μM UDP ( ii ), or [ γ 32 P]ATP and 10 μM AMP ( iii ) was added to cell cultures or to conditioned medium (Con. medium) for the indicated times, and the formation of 32 Pi (i), [ 32 P]UTP ( ii ), and [ 32 P]ADP ( iii ) were quantified by HPLC (mean ± SD from two experiments performed in triplicates). The results are expressed as percent conversion of [ 32 P]ATP to [ 32 P]-product, relative to T = 0”. The area of each radioactive peak was calculated using the HPLC analysis software provided by the manufacturer

Techniques Used: High Performance Liquid Chromatography, Incubation, Radioactivity, Software

35) Product Images from "Immunomodulating Properties of the Antibiotic Novobiocin in Human Monocytes"

Article Title: Immunomodulating Properties of the Antibiotic Novobiocin in Human Monocytes

Journal: Antimicrobial Agents and Chemotherapy

doi:

Effect of novobiocin on phosphorylation of cytosolic proteins. Monocytes (4 × 10 6 /ml) were incubated in the presence or absence of 0.5 mM novobiocin for 10 min before stimulation with LPS (100 ng/ml). After 4 h, cytosolic supernatants were prepared and incubated with [γ- 32 P]ATP for 10 min. The proteins were separated by SDS-PAGE as described in Materials and Methods. The autoradiograph is representative of three experiments.
Figure Legend Snippet: Effect of novobiocin on phosphorylation of cytosolic proteins. Monocytes (4 × 10 6 /ml) were incubated in the presence or absence of 0.5 mM novobiocin for 10 min before stimulation with LPS (100 ng/ml). After 4 h, cytosolic supernatants were prepared and incubated with [γ- 32 P]ATP for 10 min. The proteins were separated by SDS-PAGE as described in Materials and Methods. The autoradiograph is representative of three experiments.

Techniques Used: Incubation, SDS Page, Autoradiography

36) Product Images from "Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin"

Article Title: Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

Journal: Eukaryotic Cell

doi: 10.1128/EC.3.2.277-287.2004

A UMSBP monomer binds the UMS DNA. 1.0 ng of 32 P-labeled UMS DNA (lanes a and e), 1 μg of UMSBP (lanes b and f), and UMSBP with 32 P-labeled UMS DNA (lanes c, d, g, and h) were incubated in the presence of 10 mM DMP. In lanes d and h, the nucleoprotein complexes were treated first with micrococcal nuclease and subsequently with [γ- 32 P]ATP, using polynucleotide kinase, as described in Materials and Methods. The reaction products were analyzed by SDS-PAGE (10% polyacrylamide) under denaturing and reducing conditions and transferred to a nitrocellulose membrane as described in Materials and Methods. (A) 32 P-labeled UMS DNA was detected by exposure to a phosphorimaging plate. (B) The membrane was analyzed by Western blotting using anti-UMSBP antibodies and developed using an ECL protocol, as described in Materials and Methods.
Figure Legend Snippet: A UMSBP monomer binds the UMS DNA. 1.0 ng of 32 P-labeled UMS DNA (lanes a and e), 1 μg of UMSBP (lanes b and f), and UMSBP with 32 P-labeled UMS DNA (lanes c, d, g, and h) were incubated in the presence of 10 mM DMP. In lanes d and h, the nucleoprotein complexes were treated first with micrococcal nuclease and subsequently with [γ- 32 P]ATP, using polynucleotide kinase, as described in Materials and Methods. The reaction products were analyzed by SDS-PAGE (10% polyacrylamide) under denaturing and reducing conditions and transferred to a nitrocellulose membrane as described in Materials and Methods. (A) 32 P-labeled UMS DNA was detected by exposure to a phosphorimaging plate. (B) The membrane was analyzed by Western blotting using anti-UMSBP antibodies and developed using an ECL protocol, as described in Materials and Methods.

Techniques Used: Labeling, Incubation, SDS Page, Western Blot

37) Product Images from "Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis *"

Article Title: Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis *

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M405367200

Bilateral accumulation of adenyl purines Well differentiated HNE ( A ) and HBE cells ( B ), and Calu-3 cell monolayers ( C ) were rinsed and incubated in 0.3 ml of bilateral HBSS, and 200 µl were collected at 2, 30, 90, or 180 min and derivatized. Trace amounts (0.1 µCi) of [γ- 32 P]ATP or [ 3 H]adenosine were added for the indicated times to 0.3 ml of HBSS bathing the mucosal ( M ) and basolateral ( BL ) surfaces of Calu-3 cells ( C, inset ). Fluorescent and radioactive species were analyzed by HPLC, as indicated under “Experimental Procedures.” The data indicate the mean value (±S.D., n = 3) from a single experiment that was representative of two independent experiments performed under similar conditions. Mass measurements at t = 0 ( i.e. nominally purine-free HBSS) are not depicted.
Figure Legend Snippet: Bilateral accumulation of adenyl purines Well differentiated HNE ( A ) and HBE cells ( B ), and Calu-3 cell monolayers ( C ) were rinsed and incubated in 0.3 ml of bilateral HBSS, and 200 µl were collected at 2, 30, 90, or 180 min and derivatized. Trace amounts (0.1 µCi) of [γ- 32 P]ATP or [ 3 H]adenosine were added for the indicated times to 0.3 ml of HBSS bathing the mucosal ( M ) and basolateral ( BL ) surfaces of Calu-3 cells ( C, inset ). Fluorescent and radioactive species were analyzed by HPLC, as indicated under “Experimental Procedures.” The data indicate the mean value (±S.D., n = 3) from a single experiment that was representative of two independent experiments performed under similar conditions. Mass measurements at t = 0 ( i.e. nominally purine-free HBSS) are not depicted.

Techniques Used: Incubation, High Performance Liquid Chromatography

38) Product Images from "Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling"

Article Title: Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.128

Phosphorylation and activation of nSMase1 by JNK in vitro . ( a ) The serine-270 phosphorylation of nSMase1 by JNK in vitro . Mouse JNK1 was used for an in vitro kinase assay using [ γ - 32 P]-ATP and either nSMase1 (wild-type) or mutant protein (S270A) as the substrate. The reactions were incubated at 30 °C for 30 min with or without 2 ng of mouse JNK1 and in the presence (+) or absence (−) of 10 μ M SP600125. Recombinant proteins from each reaction were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Phosphorylated nSMase1 was detected by autoradiography (upper panel). The levels of substrate protein present in each reaction was determined using Coomassie Brilliant Blue R-250 staining (lower panel). ( b ) The effect of phosphorylation on the activation of nSMase1. The nSMase activity in each column is shown. After the in vitro kinase assay, the postreaction mixture was analyzed in an nSMase enzymatic assay using C 6 -NBD-sphingomyelin (black columns). White columns indicate the basal enzyme activity before the kinase assay. The basal enzyme activities of the recombinant nSMase1 (wild-type) and nSMase1 mutant (S270A) were 22.4±0.48 and 22.6±0.35 μ mol/mg/h, respectively. Column 1, nSMase1 wild-type; column 2, nSMase1 treated with JNK; column 3, nSMase1 mutant (S270A) treated with JNK; column 4, nSMase1 treated with JNK in the presence of a JNK inhibitor. The enzyme activity after JNK treatment (black column 2) was 120.1±21.4 μ mol/mg/h. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. * P
Figure Legend Snippet: Phosphorylation and activation of nSMase1 by JNK in vitro . ( a ) The serine-270 phosphorylation of nSMase1 by JNK in vitro . Mouse JNK1 was used for an in vitro kinase assay using [ γ - 32 P]-ATP and either nSMase1 (wild-type) or mutant protein (S270A) as the substrate. The reactions were incubated at 30 °C for 30 min with or without 2 ng of mouse JNK1 and in the presence (+) or absence (−) of 10 μ M SP600125. Recombinant proteins from each reaction were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Phosphorylated nSMase1 was detected by autoradiography (upper panel). The levels of substrate protein present in each reaction was determined using Coomassie Brilliant Blue R-250 staining (lower panel). ( b ) The effect of phosphorylation on the activation of nSMase1. The nSMase activity in each column is shown. After the in vitro kinase assay, the postreaction mixture was analyzed in an nSMase enzymatic assay using C 6 -NBD-sphingomyelin (black columns). White columns indicate the basal enzyme activity before the kinase assay. The basal enzyme activities of the recombinant nSMase1 (wild-type) and nSMase1 mutant (S270A) were 22.4±0.48 and 22.6±0.35 μ mol/mg/h, respectively. Column 1, nSMase1 wild-type; column 2, nSMase1 treated with JNK; column 3, nSMase1 mutant (S270A) treated with JNK; column 4, nSMase1 treated with JNK in the presence of a JNK inhibitor. The enzyme activity after JNK treatment (black column 2) was 120.1±21.4 μ mol/mg/h. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. * P

Techniques Used: Activation Assay, In Vitro, Kinase Assay, Mutagenesis, Incubation, Recombinant, Autoradiography, Staining, Activity Assay, Enzymatic Assay

39) Product Images from "Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB"

Article Title: Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.15.4599-4608.2001

MicB* autokinase activity lies in the C-terminal part of the protein and is under the control of PAS. MicB*, MicB*-N, and MicB*100LR were autophosphosphorylated with [γ- 32 P]ATP at 23°C in 48 μl of phosphorylation buffer (see Materials and Methods) containing 12 pmol of purified protein. At intervals, 16-μl aliquots were mixed with 2× loading buffer and run on SDS-12% PAGE gels for analysis as described in Materials and Methods. (C) Control in which [α- 32 P]/ATP replaced [γ- 32 P]/ATP in the reaction mixture containing MicB*.
Figure Legend Snippet: MicB* autokinase activity lies in the C-terminal part of the protein and is under the control of PAS. MicB*, MicB*-N, and MicB*100LR were autophosphosphorylated with [γ- 32 P]ATP at 23°C in 48 μl of phosphorylation buffer (see Materials and Methods) containing 12 pmol of purified protein. At intervals, 16-μl aliquots were mixed with 2× loading buffer and run on SDS-12% PAGE gels for analysis as described in Materials and Methods. (C) Control in which [α- 32 P]/ATP replaced [γ- 32 P]/ATP in the reaction mixture containing MicB*.

Techniques Used: Activity Assay, Purification, Polyacrylamide Gel Electrophoresis

40) Product Images from "The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation"

Article Title: The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation

Journal: Cell Stress & Chaperones

doi: 10.1043/1355-8145(2000)005 < 0052:THSIGS > 2.0.CO;2

Effect of geldanamycin (GA) on protein kinases involved in T-lymphocyte signaling. J32 cells (5 × 10 6 cells/mL) were incubated in the presence or absence of 1.78 μM GA at 37°C for 12 hours. Cells were serum starved during the last 4 hours of incubation and then stimulated with the T-cell receptor (TCR) ε-specific mAb OKT3 (OKT) at a final concentration of 5 μg/mL for 0 to 20 minutes as indicated. Cells were then washed and lysed in the presence of 1% Brij 98, and postnuclear supernatants of detergent extracts were prepared. In Western blot analysis (panels A, B, and D), 30 μg of postnuclear supernatant of detergent extracts were separated by 7.5% (panels A and B) or 10% (panel D) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with the respective antibodies as described in the text. In the MAPR kinase (MEK) kinase assay, 0.5 μg inactive ERK-2 was added to 5 μg of postnuclear supernatants of cell lysates and incubated in the presence of 50 μM [γ- 32 P]ATP for 20 minutes at 30°C as described in the text. (A) Effect of GA on Lck hyperphosphorylation. (B) Effect of GA on Raf-1 hyperphosphorylation. (C) Effect of GA on MEK activity as measured by the phosphorylation of inactive ERK-2. (D) Effect of GA on the endogenous ERK-2 hyperphosphorylation. Western blots and the autoradiogram are representatives of 5 independent experiments
Figure Legend Snippet: Effect of geldanamycin (GA) on protein kinases involved in T-lymphocyte signaling. J32 cells (5 × 10 6 cells/mL) were incubated in the presence or absence of 1.78 μM GA at 37°C for 12 hours. Cells were serum starved during the last 4 hours of incubation and then stimulated with the T-cell receptor (TCR) ε-specific mAb OKT3 (OKT) at a final concentration of 5 μg/mL for 0 to 20 minutes as indicated. Cells were then washed and lysed in the presence of 1% Brij 98, and postnuclear supernatants of detergent extracts were prepared. In Western blot analysis (panels A, B, and D), 30 μg of postnuclear supernatant of detergent extracts were separated by 7.5% (panels A and B) or 10% (panel D) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with the respective antibodies as described in the text. In the MAPR kinase (MEK) kinase assay, 0.5 μg inactive ERK-2 was added to 5 μg of postnuclear supernatants of cell lysates and incubated in the presence of 50 μM [γ- 32 P]ATP for 20 minutes at 30°C as described in the text. (A) Effect of GA on Lck hyperphosphorylation. (B) Effect of GA on Raf-1 hyperphosphorylation. (C) Effect of GA on MEK activity as measured by the phosphorylation of inactive ERK-2. (D) Effect of GA on the endogenous ERK-2 hyperphosphorylation. Western blots and the autoradiogram are representatives of 5 independent experiments

Techniques Used: Incubation, Concentration Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Kinase Assay, Activity Assay

41) Product Images from "Core histone N-termini play an essential role in mitotic chromosome condensation"

Article Title: Core histone N-termini play an essential role in mitotic chromosome condensation

Journal: The EMBO Journal

doi: 10.1093/emboj/19.3.379

Fig. 7. A kinase activity phosphorylating histone H3 at Ser10 was found associated with nucleosomes. ( A ) Nucleosome-arrested chromosome condensation exhibits a specific pattern of histone H3 phosphorylation. Sperm nuclei were incubated for 60 or 180 min in mitotic extract, containing either native or tailless nucleosomes, and DNA and histone H3 phosphorylation were visualized by Hoechst 33258 and anti-phosphorylated histone H3 antibody. ( B ) Native nucleosomes present in the extract decrease the degree of histone H3 phosphorylation of the chromosome condensation intermediates. Extracts were incubated for 30 min with native or tailless nucleosomes, the nucleosomes were pelleted by centrifugation and the supernatants supplemented with sperm nuclei. The control and the two nucleosome-treated extracts were incubated for 180 min at room temperature and the remodelled sperm were pelleted by centrifugation. The proteins from the pellets were run on an SDS–18% polyacrylamide gel, and phosphorylated histone H3 was detected with a specific antibody; a, b and c represent the results of three independent experiments. ( C ) Quantification of the data presented in (B). ( D ) The pelleted native nucleosomes are associated with kinase activity phosphorylating histone H3. The pellets from the control and nucleosome-treated extracts (see above) were resuspended and supplemented with GST–H3 and [γ– 32 P]ATP. After 30 min incubation, the samples were precipitated with TCA and analysed on an SDS–12% polyacrylamide gel. Left, Coomassie staining of the gel; right, autoradiography. Lane 1, protein marker; lane 2, control pellet; lane 3, pellet from the nucleosome-treated extract; lanes 4 and 5, autoradiography of lanes 3 and 4, respectively. The positions of GST–H3 and the protein markers are indicated.
Figure Legend Snippet: Fig. 7. A kinase activity phosphorylating histone H3 at Ser10 was found associated with nucleosomes. ( A ) Nucleosome-arrested chromosome condensation exhibits a specific pattern of histone H3 phosphorylation. Sperm nuclei were incubated for 60 or 180 min in mitotic extract, containing either native or tailless nucleosomes, and DNA and histone H3 phosphorylation were visualized by Hoechst 33258 and anti-phosphorylated histone H3 antibody. ( B ) Native nucleosomes present in the extract decrease the degree of histone H3 phosphorylation of the chromosome condensation intermediates. Extracts were incubated for 30 min with native or tailless nucleosomes, the nucleosomes were pelleted by centrifugation and the supernatants supplemented with sperm nuclei. The control and the two nucleosome-treated extracts were incubated for 180 min at room temperature and the remodelled sperm were pelleted by centrifugation. The proteins from the pellets were run on an SDS–18% polyacrylamide gel, and phosphorylated histone H3 was detected with a specific antibody; a, b and c represent the results of three independent experiments. ( C ) Quantification of the data presented in (B). ( D ) The pelleted native nucleosomes are associated with kinase activity phosphorylating histone H3. The pellets from the control and nucleosome-treated extracts (see above) were resuspended and supplemented with GST–H3 and [γ– 32 P]ATP. After 30 min incubation, the samples were precipitated with TCA and analysed on an SDS–12% polyacrylamide gel. Left, Coomassie staining of the gel; right, autoradiography. Lane 1, protein marker; lane 2, control pellet; lane 3, pellet from the nucleosome-treated extract; lanes 4 and 5, autoradiography of lanes 3 and 4, respectively. The positions of GST–H3 and the protein markers are indicated.

Techniques Used: Activity Assay, Incubation, Centrifugation, Staining, Autoradiography, Marker

42) Product Images from "Transcriptional Regulation of the phoPR Operon in Bacillus subtilis"

Article Title: Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.4.1182-1190.2004

Production and purification of PhoP-His 6 and PhoR231-His 6 and phosphorylation assay. (A) E. coli BL21(λD3) with pET-PhoP or pET-PhoR231 was grown in LB, and PhoP-His 6 and PhoR231-His 6 production was induced with IPTG. Purified proteins were analyzed by SDS-PAGE, and the gel was stained with GelCode blue stain reagent. An amount of 1 μg of protein was loaded in lanes 4 and 6, and 10 μg of protein was loaded in lanes 2, 3, and 5. Shown are prestained protein standards (lane 1), supernatant fraction of the whole-cell sonication lysate from the IPTG-induced E. coli BL21(λD3) carrying pET-PhoP (lane 3) or pET-PhoR231 (lane 5), and eluates of PhoP-His 6 (lane 4) and PhoR231-His 6 (lane 6) by 30 to 300 mM imidazole gradient. Supernatant fraction of the whole-cell sonication lysate from the noninduced E. coli BL21(λD3) carrying pET-PhoP (lane 2) was the negative control. (B) Phosphorylation of PhoP-His 6 by PhoR231-His 6 in the presence of ATP. PhoR231 (8 μM) was incubated with 40 μCi of [γ- 32 P]ATP isotope in the absence (lane 1) or the presence (lanes 2 and 3) of 24 μM PhoP for 5 min (lane 1), 10 min (lane 2), and 20 min (lane 3) at RT. The samples were subjected to SDS-PAGE and, after being dried, the gel was exposed to an X-ray film.
Figure Legend Snippet: Production and purification of PhoP-His 6 and PhoR231-His 6 and phosphorylation assay. (A) E. coli BL21(λD3) with pET-PhoP or pET-PhoR231 was grown in LB, and PhoP-His 6 and PhoR231-His 6 production was induced with IPTG. Purified proteins were analyzed by SDS-PAGE, and the gel was stained with GelCode blue stain reagent. An amount of 1 μg of protein was loaded in lanes 4 and 6, and 10 μg of protein was loaded in lanes 2, 3, and 5. Shown are prestained protein standards (lane 1), supernatant fraction of the whole-cell sonication lysate from the IPTG-induced E. coli BL21(λD3) carrying pET-PhoP (lane 3) or pET-PhoR231 (lane 5), and eluates of PhoP-His 6 (lane 4) and PhoR231-His 6 (lane 6) by 30 to 300 mM imidazole gradient. Supernatant fraction of the whole-cell sonication lysate from the noninduced E. coli BL21(λD3) carrying pET-PhoP (lane 2) was the negative control. (B) Phosphorylation of PhoP-His 6 by PhoR231-His 6 in the presence of ATP. PhoR231 (8 μM) was incubated with 40 μCi of [γ- 32 P]ATP isotope in the absence (lane 1) or the presence (lanes 2 and 3) of 24 μM PhoP for 5 min (lane 1), 10 min (lane 2), and 20 min (lane 3) at RT. The samples were subjected to SDS-PAGE and, after being dried, the gel was exposed to an X-ray film.

Techniques Used: Purification, Phosphorylation Assay, Positron Emission Tomography, SDS Page, Staining, Sonication, Negative Control, Incubation

43) Product Images from "Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide"

Article Title: Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

Journal: Journal of Virology

doi:

In vitro phosphorylation of NS1. Phosphatase-treated or native wild-type NS1 was incubated with fractionated cell extract or commercially available protein kinases (PK) in the presence of [γ- 32 P]ATP, immunoprecipitated with αNS N , and revealed by autoradiography after SDS-PAGE. HeLa S100 cell extract was fractionated by chromatography on a phosphocellulose P11 column (Whatman) into P1 (150 mM NaCl flowthrough), P2 (150 to 400 mM NaCl elution), and P3 (400 to 1,000 mM NaCl elution) fractions. cdc2, cdc2 complex. Lanes: 1 to 7 and 9, dephosphorylated NS1 used as a substrate; 8, native NS1 used as a substrate; 1, protein kinases from fraction P1; 2, protein kinases from fraction P2; 3, protein kinases from fraction P3; 4 to 7, commercially available protein kinases (cdc2 complex, CKII, PKA, and PKC, respectively); 8 and 9, protein kinases from HeLa cell replication extracts. The heavily labeled 32-kDa protein in lane 6 probably corresponds to the autophosphorylated catalytic subunit of PKA present in large amounts in the reaction.
Figure Legend Snippet: In vitro phosphorylation of NS1. Phosphatase-treated or native wild-type NS1 was incubated with fractionated cell extract or commercially available protein kinases (PK) in the presence of [γ- 32 P]ATP, immunoprecipitated with αNS N , and revealed by autoradiography after SDS-PAGE. HeLa S100 cell extract was fractionated by chromatography on a phosphocellulose P11 column (Whatman) into P1 (150 mM NaCl flowthrough), P2 (150 to 400 mM NaCl elution), and P3 (400 to 1,000 mM NaCl elution) fractions. cdc2, cdc2 complex. Lanes: 1 to 7 and 9, dephosphorylated NS1 used as a substrate; 8, native NS1 used as a substrate; 1, protein kinases from fraction P1; 2, protein kinases from fraction P2; 3, protein kinases from fraction P3; 4 to 7, commercially available protein kinases (cdc2 complex, CKII, PKA, and PKC, respectively); 8 and 9, protein kinases from HeLa cell replication extracts. The heavily labeled 32-kDa protein in lane 6 probably corresponds to the autophosphorylated catalytic subunit of PKA present in large amounts in the reaction.

Techniques Used: In Vitro, Incubation, Immunoprecipitation, Autoradiography, SDS Page, Chromatography, Labeling

Effect of dephosphorylation on ATPase activity of NS1. Released 32 P i was determined by scintillation counting after incubation of [γ- 32 P]ATP with native or dephosphorylated NS1. Average values from multiple assays using different sucrose gradient fractions are shown with their standard deviation bars for two independent NS1 O preparations (#1 and #2). The NS1 mutant K405R served as a negative control. Data are expressed relative to the ATPase activity of native wild-type NS1 (wt P ).
Figure Legend Snippet: Effect of dephosphorylation on ATPase activity of NS1. Released 32 P i was determined by scintillation counting after incubation of [γ- 32 P]ATP with native or dephosphorylated NS1. Average values from multiple assays using different sucrose gradient fractions are shown with their standard deviation bars for two independent NS1 O preparations (#1 and #2). The NS1 mutant K405R served as a negative control. Data are expressed relative to the ATPase activity of native wild-type NS1 (wt P ).

Techniques Used: De-Phosphorylation Assay, Activity Assay, Incubation, Standard Deviation, Mutagenesis, Negative Control

44) Product Images from "Identification of Specific Molecular Structures of Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological Effects on Vascular Endothelial Cells"

Article Title: Identification of Specific Molecular Structures of Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological Effects on Vascular Endothelial Cells

Journal: Journal of Virology

doi:

Effect of Tat molecules on PI 3-kinase in endothelial cells. Quiescent, confluent ECs were stimulated with Tat molecules (20 ng/ml) for 15 min at 37°C. The PI 3-kinase assay was performed on immune complexes done with MAb antiphosphotyrosine (anti-PY) antibodies from lysates of EC in the presence of 40 μM ATP, 50 μCi of [γ- 32 P]ATP, and 50 μg of a presonicated mixture of phosphatidylinositol-4,5-bisphosphate and phosphatidylserine per ml in 25 mM HEPES (pH 7.4)–1 mM EGTA. The extracted lipids were separated by thin-layer chromatography and visualized by autoradiography. PIP 3 , phosphatidylinositol-3,4-bisphosphate; Prot-A, a PI 3-kinase assay done on protein A alone. Spots corresponding to PIP 3 were recovered and counted ( n = 3): control, 420 ± 132 cpm; Tat 86 , 1,342 ± 231; Tat R78K/D80E, 1,280 ± 280; Tat R49G/K50I/R52L/R53I, 750 ± 201. I.P., immunoprecipitate.
Figure Legend Snippet: Effect of Tat molecules on PI 3-kinase in endothelial cells. Quiescent, confluent ECs were stimulated with Tat molecules (20 ng/ml) for 15 min at 37°C. The PI 3-kinase assay was performed on immune complexes done with MAb antiphosphotyrosine (anti-PY) antibodies from lysates of EC in the presence of 40 μM ATP, 50 μCi of [γ- 32 P]ATP, and 50 μg of a presonicated mixture of phosphatidylinositol-4,5-bisphosphate and phosphatidylserine per ml in 25 mM HEPES (pH 7.4)–1 mM EGTA. The extracted lipids were separated by thin-layer chromatography and visualized by autoradiography. PIP 3 , phosphatidylinositol-3,4-bisphosphate; Prot-A, a PI 3-kinase assay done on protein A alone. Spots corresponding to PIP 3 were recovered and counted ( n = 3): control, 420 ± 132 cpm; Tat 86 , 1,342 ± 231; Tat R78K/D80E, 1,280 ± 280; Tat R49G/K50I/R52L/R53I, 750 ± 201. I.P., immunoprecipitate.

Techniques Used: Kinase Assay, Thin Layer Chromatography, Autoradiography

45) Product Images from "Expression, purification and characterization of methyl DNA binding protein from Bombyx mori"

Article Title: Expression, purification and characterization of methyl DNA binding protein from Bombyx mori

Journal: Journal of Insect Science

doi:

Phosphorylation of bMBD2/3 in vitro . The reaction mixture containing bMBD2/3 proteins and protein kinases [lane 1, Protein kinase A; lane 2, protein kinase C; lane 3, Ca/CaM dependent protein kinaseII; lane 4, brain extract; lane 5 brain extract (-bMBD2/3)] were incubated with [γ- 32 P]ATP. The samples were treated with SDS–sample buffer. After electrophoresis, the gel was analyzed using a bioimaging analyzer (upper lanes). The bands stained with Coomassie Brilliant Blue after electrophoresis (lower lanes).
Figure Legend Snippet: Phosphorylation of bMBD2/3 in vitro . The reaction mixture containing bMBD2/3 proteins and protein kinases [lane 1, Protein kinase A; lane 2, protein kinase C; lane 3, Ca/CaM dependent protein kinaseII; lane 4, brain extract; lane 5 brain extract (-bMBD2/3)] were incubated with [γ- 32 P]ATP. The samples were treated with SDS–sample buffer. After electrophoresis, the gel was analyzed using a bioimaging analyzer (upper lanes). The bands stained with Coomassie Brilliant Blue after electrophoresis (lower lanes).

Techniques Used: In Vitro, Chick Chorioallantoic Membrane Assay, Incubation, Electrophoresis, Staining

46) Product Images from "GSTT2 promoter polymorphisms and colorectal cancer risk"

Article Title: GSTT2 promoter polymorphisms and colorectal cancer risk

Journal: BMC Cancer

doi: 10.1186/1471-2407-7-16

EMSA with HeLa nuclear extracts using -537G and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.
Figure Legend Snippet: EMSA with HeLa nuclear extracts using -537G and -537A oligonucleotides . Binding activities of [γ- 32 P] ATP-labeled -537G (lane 1–6) and -537A (lane 7–12) oligonucleotides. The assay was performed in the presence (+) or absence (-) of HeLa nuclear extracts. Unlabeled -537G or -537A oligonucleotides were used in competition assays. Each binding reaction contained 5 mg of HeLa nuclear extracts and labeled -537G (lanes 2–6) or -537A (lanes 8–12) oligonucleotides. Excess unlabeled oligonucleotides (10-, 50- and 100-fold) were included in the binding reactions as competitors (Lanes 3–5 and 9–11, respectively). In addition, we added a 100-fold excess of unlabeled -537A and -537G oligonucleotides to compete with -537G (Lane 6) and -537A (Lane 12) oligonucleotides. The binding activity of -537G was unaffected, even in the presence of a 100-fold excess of -537A competitor (lane 6). However, the -537A oligonucleotide could not bind transcription factor (lane 12), and displayed no band in the presence of a 100-fold excess of -537G probe.

Techniques Used: Binding Assay, Labeling, Activity Assay

47) Product Images from "The RNA-Binding Protein KSRP Promotes Decay of ?-Catenin mRNA and Is Inactivated by PI3K-AKT Signaling"

Article Title: The RNA-Binding Protein KSRP Promotes Decay of ?-Catenin mRNA and Is Inactivated by PI3K-AKT Signaling

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0050005

KSRP Is Phosphorylated by AKT in Serine 193 (A) Immunoprecipitation of β-catenin RNA-containing ribonucleoprotein complexes. The proteins were immunoprecipitated from αT3-1 total cell extracts using the indicated antibodies. RNA was extracted from the immune complexes and analyzed by RT-PCR as indicated. (B) Either highly purified recombinant human KSRP (lanes 1 and 2) or affinity-purified GST-HuR (lanes 3 and 4) was incubated with either active recombinant AKT2 in kinase buffer or buffer alone in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE, blotted to nylon membranes, and detected by autoradiography (top panel). Immunoblot analysis of the above kinase reactions with either anti-KSRP (middle panel) or anti-GST (bottom panel) antibodies. (C) Schematic representation of KSRP and GST-fused deletion mutants. Asterisks indicate the position of a putative AKT phosphorylation site (left). AKT2 kinase assays were performed using the indicated recombinant protein substrates (middle and right). Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2. (D) In vivo [ 32 P]orthophosphate metabolic labeling of HeLa cells transiently cotransfected with either FLAG-KSRP plus empty pcDNA3, or FLAG-KSRP plus pCMV–myr-AKT1, or FLAG-KSRP(S193A) plus pCMV–myr-AKT1. Aliquots of the lysates were immunoprecipitated with protein A–protein G–Sepharose–bound mouse control IgG (cIgG) or anti-FLAG antibody, separated by SDS-PAGE, and autoradiographed (top panel). Immunoblot analysis of the above in vivo phosphorylation reactions with anti-FLAG antibody (bottom panel). Arrows point to the position of transfected KSRP. (E) MS-MS fragmentation spectrum of the tryptic peptide SV[pS]LTGAPESVQK of KSRP showing phosphorylation of S193. (F) AKT2 kinase assays using the indicated recombinant protein substrates. Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2.
Figure Legend Snippet: KSRP Is Phosphorylated by AKT in Serine 193 (A) Immunoprecipitation of β-catenin RNA-containing ribonucleoprotein complexes. The proteins were immunoprecipitated from αT3-1 total cell extracts using the indicated antibodies. RNA was extracted from the immune complexes and analyzed by RT-PCR as indicated. (B) Either highly purified recombinant human KSRP (lanes 1 and 2) or affinity-purified GST-HuR (lanes 3 and 4) was incubated with either active recombinant AKT2 in kinase buffer or buffer alone in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE, blotted to nylon membranes, and detected by autoradiography (top panel). Immunoblot analysis of the above kinase reactions with either anti-KSRP (middle panel) or anti-GST (bottom panel) antibodies. (C) Schematic representation of KSRP and GST-fused deletion mutants. Asterisks indicate the position of a putative AKT phosphorylation site (left). AKT2 kinase assays were performed using the indicated recombinant protein substrates (middle and right). Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2. (D) In vivo [ 32 P]orthophosphate metabolic labeling of HeLa cells transiently cotransfected with either FLAG-KSRP plus empty pcDNA3, or FLAG-KSRP plus pCMV–myr-AKT1, or FLAG-KSRP(S193A) plus pCMV–myr-AKT1. Aliquots of the lysates were immunoprecipitated with protein A–protein G–Sepharose–bound mouse control IgG (cIgG) or anti-FLAG antibody, separated by SDS-PAGE, and autoradiographed (top panel). Immunoblot analysis of the above in vivo phosphorylation reactions with anti-FLAG antibody (bottom panel). Arrows point to the position of transfected KSRP. (E) MS-MS fragmentation spectrum of the tryptic peptide SV[pS]LTGAPESVQK of KSRP showing phosphorylation of S193. (F) AKT2 kinase assays using the indicated recombinant protein substrates. Arrows point to the position of each phosphorylated protein; the asterisk marks the position of phosphorylated AKT2.

Techniques Used: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Purification, Recombinant, Affinity Purification, Incubation, Labeling, SDS Page, Autoradiography, In Vivo, Transfection, Mass Spectrometry

PI3K-AKT Signaling Stabilizes β-Catenin mRNA and Increases Its Expression (A) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of LY, control), DMSO plus LiCl, or LY (25 μM for 16 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of triciribine, control), DMSO plus LiCl (20 mM for 6 h), or triciribine (1 μM for 1 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (C) In vitro RNA degradation assays using S100s from either LiCl-treated mock αT3-1 or LiCl-treated αT3-1 cells expressing the AKT dominant negative mutant AKT1(K179M) (αT3-1–AKTDN). Internally 32 P-labeled and capped RNA substrates were incubated with S100s for the indicated times and their decay analyzed as described in Materials and Methods. (D) Either mock αT3-1 or αT3-1–myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–myrAKT1 cells. (F) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–myrAKT1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (G) Immunoblot analysis of either S100 or nuclear extracts from the indicated cell lines with anti- β-catenin, α-tubulin, and HDAC2 antibodies.
Figure Legend Snippet: PI3K-AKT Signaling Stabilizes β-Catenin mRNA and Increases Its Expression (A) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of LY, control), DMSO plus LiCl, or LY (25 μM for 16 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells treated with either DMSO (the solvent of triciribine, control), DMSO plus LiCl (20 mM for 6 h), or triciribine (1 μM for 1 h) plus LiCl. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (C) In vitro RNA degradation assays using S100s from either LiCl-treated mock αT3-1 or LiCl-treated αT3-1 cells expressing the AKT dominant negative mutant AKT1(K179M) (αT3-1–AKTDN). Internally 32 P-labeled and capped RNA substrates were incubated with S100s for the indicated times and their decay analyzed as described in Materials and Methods. (D) Either mock αT3-1 or αT3-1–myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either mock αT3-1 or αT3-1–myrAKT1 cells. (F) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either mock αT3-1 or αT3-1–myrAKT1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (G) Immunoblot analysis of either S100 or nuclear extracts from the indicated cell lines with anti- β-catenin, α-tubulin, and HDAC2 antibodies.

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, In Vitro, Dominant Negative Mutation, Labeling, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Reverse Transcription Polymerase Chain Reaction

Insulin Stabilizes β-Catenin mRNA and Increases Its Expression (A) HIRc-B cells were treated for 1 h with DMSO (control), insulin (10 −6 M) plus DMSO, or LY (25 μM for 16 h) plus insulin. Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in control HIRc-B cells and in HIRc-B treated with either insulin or LY plus insulin. (C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in control HIRc-B cells, in HIRc-B treated with either insulin or LY plus insulin. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (D) Immunoblot analysis of either S100 or nuclear extracts from either control- or insulin-treated HIRc-B cells with anti–β-catenin, β-actin, and HDAC2 antibodies. The amount of each band was quantitated by densitometry and insulin was found to increase β-catenin expression by 3.2- and 2.1-fold over the control in S100 and nuclear extracts, respectively. (E) HIRc-B cells were maintained for 16 h in DMEM containing 0.1% FCS; then either PBS (control) or insulin (10 −6 M) was added for 1 h. Cultures were then treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared, and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. β-Actin immunoblotting was used to verify the equal protein loading.
Figure Legend Snippet: Insulin Stabilizes β-Catenin mRNA and Increases Its Expression (A) HIRc-B cells were treated for 1 h with DMSO (control), insulin (10 −6 M) plus DMSO, or LY (25 μM for 16 h) plus insulin. Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30 °C) with histone 2B (H2B) in kinase buffer in the presence of [γ- 32 P]ATP. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of β-catenin and β2-MG, monitored by RT-PCR, in control HIRc-B cells and in HIRc-B treated with either insulin or LY plus insulin. (C) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in control HIRc-B cells, in HIRc-B treated with either insulin or LY plus insulin. Total RNA was isolated at the indicated times after the addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate. (D) Immunoblot analysis of either S100 or nuclear extracts from either control- or insulin-treated HIRc-B cells with anti–β-catenin, β-actin, and HDAC2 antibodies. The amount of each band was quantitated by densitometry and insulin was found to increase β-catenin expression by 3.2- and 2.1-fold over the control in S100 and nuclear extracts, respectively. (E) HIRc-B cells were maintained for 16 h in DMEM containing 0.1% FCS; then either PBS (control) or insulin (10 −6 M) was added for 1 h. Cultures were then treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared, and the levels of β-catenin quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. β-Actin immunoblotting was used to verify the equal protein loading.

Techniques Used: Expressing, Immunoprecipitation, Incubation, Labeling, SDS Page, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

48) Product Images from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"

Article Title: Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-8-28

Insulin stabilizes a set of KSRP-interacting mRNAs. (A) Serum-starved HIRc-B cells were treated for 1 h with either PBS (control) or insulin (10 -6 M). Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) In vitro RNA degradation assays using S100 extracts from either control or insulin (10 -6 M)-treated HIRc-B cells. Internally 32 P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with S100 extracts for the indicated times and their decay analyzed as described in Methods.
Figure Legend Snippet: Insulin stabilizes a set of KSRP-interacting mRNAs. (A) Serum-starved HIRc-B cells were treated for 1 h with either PBS (control) or insulin (10 -6 M). Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) In vitro RNA degradation assays using S100 extracts from either control or insulin (10 -6 M)-treated HIRc-B cells. Internally 32 P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with S100 extracts for the indicated times and their decay analyzed as described in Methods.

Techniques Used: Immunoprecipitation, Incubation, Labeling, SDS Page, Autoradiography, In Vitro

PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression. (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 7 .
Figure Legend Snippet: PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression. (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 7 .

Techniques Used: Expressing, Immunoprecipitation, Incubation, Labeling, SDS Page, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Quantitation Assay

49) Product Images from "Both XPD alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients"

Article Title: Both XPD alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20091892

Transcription activity of the rIIHs. (A) Basal transcription activity of the rIIHs. The purified rIIHs were added to an in vitro reconstituted transcription system lacking TFIIH. The length of the corresponding transcript is indicated on the left side. The transcription activity of all variants was assessed using increasing amounts of rIIHs for 30 min. The signals were quantified and plotted in arbitrary units (au). The results are representative of three independent experiments. The complete gel is shown in Fig. S2 A. (B) Phosphorylation of the RNA polymerase II during in vitro reconstituted transcription assays. The RNA pol II kinase activity of low salt immunopurifed rIIHs was analyzed in an in vitro assay containing all the basal transcription factors and the AdMLP. Arrows indicate hypo (IIA) and hyper (IIO) phosphorylated forms of RNA pol II. The results are representative of two independent experiments. The complete gel is shown in Fig. S2 B. (C) In vitro phosphorylation of the GST-CTD fusion protein was performed with equal amounts of rIIHs in the presence of 0.14 µM [γ- 32 P] ATP. Coomassie blue–stained gel (staining; top) and autoradiography (autoradio; bottom) of the incubated fractions are shown. The results are representative of three independent experiments. The complete gels are shown in Fig. S2 C. (D) In vitro reconstituted transcription assays were performed following the protocol scheme. rIIH XPD/R683W, rIIH XPD/Q452X, /199insPP, or /I455del were preincubated either alone or in combination, in the presence of RNA pol II, the general transcription factors (GTF), the AdMLP template, ATP, and CTP. 15 min later, the transcription process was initiated by addition of GTP and UTP. The reactions were performed for 15 and 30 min. The signals of three independent experiments were then quantified and plotted in arbitrary units (au).
Figure Legend Snippet: Transcription activity of the rIIHs. (A) Basal transcription activity of the rIIHs. The purified rIIHs were added to an in vitro reconstituted transcription system lacking TFIIH. The length of the corresponding transcript is indicated on the left side. The transcription activity of all variants was assessed using increasing amounts of rIIHs for 30 min. The signals were quantified and plotted in arbitrary units (au). The results are representative of three independent experiments. The complete gel is shown in Fig. S2 A. (B) Phosphorylation of the RNA polymerase II during in vitro reconstituted transcription assays. The RNA pol II kinase activity of low salt immunopurifed rIIHs was analyzed in an in vitro assay containing all the basal transcription factors and the AdMLP. Arrows indicate hypo (IIA) and hyper (IIO) phosphorylated forms of RNA pol II. The results are representative of two independent experiments. The complete gel is shown in Fig. S2 B. (C) In vitro phosphorylation of the GST-CTD fusion protein was performed with equal amounts of rIIHs in the presence of 0.14 µM [γ- 32 P] ATP. Coomassie blue–stained gel (staining; top) and autoradiography (autoradio; bottom) of the incubated fractions are shown. The results are representative of three independent experiments. The complete gels are shown in Fig. S2 C. (D) In vitro reconstituted transcription assays were performed following the protocol scheme. rIIH XPD/R683W, rIIH XPD/Q452X, /199insPP, or /I455del were preincubated either alone or in combination, in the presence of RNA pol II, the general transcription factors (GTF), the AdMLP template, ATP, and CTP. 15 min later, the transcription process was initiated by addition of GTP and UTP. The reactions were performed for 15 and 30 min. The signals of three independent experiments were then quantified and plotted in arbitrary units (au).

Techniques Used: Activity Assay, Purification, In Vitro, Staining, Autoradiography, Incubation

50) Product Images from "Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain"

Article Title: Novel Role for an HPt Domain in Stabilizing the Phosphorylated State of a Response Regulator Domain

Journal: Journal of Bacteriology

doi:

Phosphorylation of response regulator domains. Phosphorylation reaction mixtures (30 μl) contained 4 μM GST-SLN1-HK and 11 μM [γ- 32 P]ATP in the absence and presence of 8 μM of the response regulator domains as indicated. Reaction mixtures were incubated for 60 min at room temperature, and reactions were stopped by the addition of 10 μl of 4× stop buffer. Reaction products were separated on an SDS-15% polyacrylamide gel. Immediately following SDS-polyacrylamide gel electrophoresis, the wet gel was wrapped in plastic film and subjected to autoradiography at −80°C.
Figure Legend Snippet: Phosphorylation of response regulator domains. Phosphorylation reaction mixtures (30 μl) contained 4 μM GST-SLN1-HK and 11 μM [γ- 32 P]ATP in the absence and presence of 8 μM of the response regulator domains as indicated. Reaction mixtures were incubated for 60 min at room temperature, and reactions were stopped by the addition of 10 μl of 4× stop buffer. Reaction products were separated on an SDS-15% polyacrylamide gel. Immediately following SDS-polyacrylamide gel electrophoresis, the wet gel was wrapped in plastic film and subjected to autoradiography at −80°C.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

51) Product Images from "Protein Kinase SGK Mediates Survival Signals by Phosphorylating the Forkhead Transcription Factor FKHRL1 (FOXO3a)"

Article Title: Protein Kinase SGK Mediates Survival Signals by Phosphorylating the Forkhead Transcription Factor FKHRL1 (FOXO3a)

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.3.952-965.2001

SGK phosphorylates FKHRL1 in vitro. (A) HEK 293 cells were transfected with WT SGK, a KN mutant of SGK (K127Q), or a CA mutant of SGK (S422D) and stimulated with insulin or IGF-I for 15 min. SGK was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 (upper panels). The total level of GST-FKHRL1 was analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panels). (B) HEK 293 cells were transfected with a CA mutant of Akt. Akt was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (upper panel). Purified CA SGK was obtained from Upstate Biotechnology and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (middle panel). The total levels of GST-FKHRL1 were analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panel). CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). The numbers represent the percentage of phosphorylation of FKHRL1 mutants compared to wild-type FKHRL1. (C) Extracts obtained for panel B with WT-FKHRL1 were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, and phospho-S315 or with the antibody directed against total FKHRL1.
Figure Legend Snippet: SGK phosphorylates FKHRL1 in vitro. (A) HEK 293 cells were transfected with WT SGK, a KN mutant of SGK (K127Q), or a CA mutant of SGK (S422D) and stimulated with insulin or IGF-I for 15 min. SGK was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 (upper panels). The total level of GST-FKHRL1 was analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panels). (B) HEK 293 cells were transfected with a CA mutant of Akt. Akt was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (upper panel). Purified CA SGK was obtained from Upstate Biotechnology and incubated in the presence of [γ- 32 P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (middle panel). The total levels of GST-FKHRL1 were analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panel). CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). The numbers represent the percentage of phosphorylation of FKHRL1 mutants compared to wild-type FKHRL1. (C) Extracts obtained for panel B with WT-FKHRL1 were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, and phospho-S315 or with the antibody directed against total FKHRL1.

Techniques Used: In Vitro, Transfection, Mutagenesis, Immunoprecipitation, Incubation, Purification, CTL Assay

52) Product Images from "Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A"

Article Title: Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A

Journal: Eukaryotic Cell

doi: 10.1128/EC.3.5.1147-1153.2004

PKA and Crz1p interact in vivo and in vitro. (A) Crz1p is phosphorylated by Tpk1p in vitro and can subsequently be dephosphorylated by calcineurin. Recombinant GST-Crz1p and GST-Tpk1p were incubated with ATP and [γ- 32 P]ATP for 40 min at 30°C,
Figure Legend Snippet: PKA and Crz1p interact in vivo and in vitro. (A) Crz1p is phosphorylated by Tpk1p in vitro and can subsequently be dephosphorylated by calcineurin. Recombinant GST-Crz1p and GST-Tpk1p were incubated with ATP and [γ- 32 P]ATP for 40 min at 30°C,

Techniques Used: In Vivo, In Vitro, Recombinant, Incubation

53) Product Images from "Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs"

Article Title: Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf639

Fig. 3 . Mapping of the site in hnRNP A0 phosphorylated by MAPKAP-K2. ( A ) Purified GST–hnRNP A0 was phosphorylated for 60 min in vitro with 150 U/ml of MAPKAP-K2 and 0.1 mM [γ- 32 P]ATP, and subjected to SDS–PAGE. The band corresponding to GST–hnRNP A0 was excised, digested with trypsin and the resulting peptides separated by reverse phase hydrophobic interaction chromatography on a C 18 column equilibrated in 0.1% trifluoroacetic acid. 32 P-radioactivity is shown by the full line, and the acetonitrile gradient by the broken line. ( B ).
Figure Legend Snippet: Fig. 3 . Mapping of the site in hnRNP A0 phosphorylated by MAPKAP-K2. ( A ) Purified GST–hnRNP A0 was phosphorylated for 60 min in vitro with 150 U/ml of MAPKAP-K2 and 0.1 mM [γ- 32 P]ATP, and subjected to SDS–PAGE. The band corresponding to GST–hnRNP A0 was excised, digested with trypsin and the resulting peptides separated by reverse phase hydrophobic interaction chromatography on a C 18 column equilibrated in 0.1% trifluoroacetic acid. 32 P-radioactivity is shown by the full line, and the acetonitrile gradient by the broken line. ( B ).

Techniques Used: Purification, In Vitro, SDS Page, Hydrophobic Interaction Chromatography, Radioactivity

Fig. 2 . MAPKAP-K2 phosphorylates two major proteins binding to the TNF-α ARE. Untreated RAW cell extracts were incubated with a biotin ylated RNA oligonucleotide corresponding to the TNF-α ARE. RNA-binding proteins were then phosphorylated in the absence or presence of 1 or 10 U/ml of MAPKAP-K2 and 20 nM [γ- 32 P]ATP (lanes 1–4). Proteins were then separated by SDS–PAGE, transferred to nitrocellulose membranes and autoradiographed.
Figure Legend Snippet: Fig. 2 . MAPKAP-K2 phosphorylates two major proteins binding to the TNF-α ARE. Untreated RAW cell extracts were incubated with a biotin ylated RNA oligonucleotide corresponding to the TNF-α ARE. RNA-binding proteins were then phosphorylated in the absence or presence of 1 or 10 U/ml of MAPKAP-K2 and 20 nM [γ- 32 P]ATP (lanes 1–4). Proteins were then separated by SDS–PAGE, transferred to nitrocellulose membranes and autoradiographed.

Techniques Used: Binding Assay, Incubation, RNA Binding Assay, SDS Page

54) Product Images from "Divergence of Chemical Function in the Alkaline Phosphatase Superfamily: Structure and Mechanism of the P-C Bond Cleaving Enzyme Phosphonoacetate Hydrolase"

Article Title: Divergence of Chemical Function in the Alkaline Phosphatase Superfamily: Structure and Mechanism of the P-C Bond Cleaving Enzyme Phosphonoacetate Hydrolase

Journal: Biochemistry

doi: 10.1021/bi200165h

SDS-PAGE analysis of PAc hydrolase autophosphorylation with [γ 32 P]ATP. The top panel is the Coomassie blue stained gel and the bottom panel is the audioradiograph of the gel. Lane 1: Protein molecular weight standards. Lane 2: The reaction of
Figure Legend Snippet: SDS-PAGE analysis of PAc hydrolase autophosphorylation with [γ 32 P]ATP. The top panel is the Coomassie blue stained gel and the bottom panel is the audioradiograph of the gel. Lane 1: Protein molecular weight standards. Lane 2: The reaction of

Techniques Used: SDS Page, Staining, Molecular Weight

55) Product Images from "Characterization of a Selective Inhibitor of Inositol Hexakisphosphate Kinases: USE IN DEFINING BIOLOGICAL ROLES AND METABOLIC RELATIONSHIPS OF INOSITOL PYROPHOSPHATES*S⃞"

Article Title: Characterization of a Selective Inhibitor of Inositol Hexakisphosphate Kinases: USE IN DEFINING BIOLOGICAL ROLES AND METABOLIC RELATIONSHIPS OF INOSITOL PYROPHOSPHATES*S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M900752200

Vip/PPIP5Ks are not inhibited by TNP. 100 ng of purified Vip2 fragment (amino acids 1–365 from the human Vip2) was preincubated with vehicle (DMSO) or 10 μ m TNP for 30 min on ice. Following the pretreatment, [γ- 32 P]ATP and 100
Figure Legend Snippet: Vip/PPIP5Ks are not inhibited by TNP. 100 ng of purified Vip2 fragment (amino acids 1–365 from the human Vip2) was preincubated with vehicle (DMSO) or 10 μ m TNP for 30 min on ice. Following the pretreatment, [γ- 32 P]ATP and 100

Techniques Used: Purification

56) Product Images from "Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein"

Article Title: Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.18.6592-6604.2002

) and from E. coli were tested for comparison. (A) Wild-type (WT) E1 and mutated E1 P479S protein were analyzed by SDS-PAGE and stained with Coomassie blue (left) and also by Western blot with monoclonal antibody against the epitope tag (right). (B) Autoradiogram of ATPase assays. The positions of [γ- 32 P]ATP and free 32 P i are marked. (C) Unwinding of ori plasmid p7874-99 as revealed in an ethidium bromide-stained chloroquine-agarose gel. Lane 1, supercoiled input DNA (*) in reaction buffer. Lane 2, reaction without E1. Lane 3, reaction with wild-type E1 purified from E. coli . U-form DNA is marked (
Figure Legend Snippet: ) and from E. coli were tested for comparison. (A) Wild-type (WT) E1 and mutated E1 P479S protein were analyzed by SDS-PAGE and stained with Coomassie blue (left) and also by Western blot with monoclonal antibody against the epitope tag (right). (B) Autoradiogram of ATPase assays. The positions of [γ- 32 P]ATP and free 32 P i are marked. (C) Unwinding of ori plasmid p7874-99 as revealed in an ethidium bromide-stained chloroquine-agarose gel. Lane 1, supercoiled input DNA (*) in reaction buffer. Lane 2, reaction without E1. Lane 3, reaction with wild-type E1 purified from E. coli . U-form DNA is marked (

Techniques Used: SDS Page, Staining, Western Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Purification

57) Product Images from "Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *"

Article Title: Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.144220

Plk1 phosphorylates ASAP on S289 in vitro . A , purified, recombinant GST-ASAP, α-casein, or GST was incubated with purified His 6 -tagged wild-type Plk1 (His 6 -Plk1-WT) or the kinase-dead mutant (N281A) (His 6 -Plk1-KD) and [γ- 32 P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography ( upper panel ); the gel was also stained with Coomassie Blue ( lower panel ). B , purified GST, GST-ASAP fragments G1–G6 or GST-ASAP were used in a kinase assay with His 6 -Plk1-WT as described in A and analyzed by SDS-PAGE and autoradiography ( upper panel ); the Coomassie Blue-stained gel is shown in the lower panel. C , in vitro kinase assays were performed using purified GST-ASAP, GST-ASAP mutants (S289A or S369A/S370A), or GST as described in A and analyzed by SDS-PAGE and autoradiography ( upper panel ); the Coomassie Blue-stained gel is shown in the lower panel .
Figure Legend Snippet: Plk1 phosphorylates ASAP on S289 in vitro . A , purified, recombinant GST-ASAP, α-casein, or GST was incubated with purified His 6 -tagged wild-type Plk1 (His 6 -Plk1-WT) or the kinase-dead mutant (N281A) (His 6 -Plk1-KD) and [γ- 32 P]ATP. The kinase reaction mixtures were then analyzed by SDS-PAGE and autoradiography ( upper panel ); the gel was also stained with Coomassie Blue ( lower panel ). B , purified GST, GST-ASAP fragments G1–G6 or GST-ASAP were used in a kinase assay with His 6 -Plk1-WT as described in A and analyzed by SDS-PAGE and autoradiography ( upper panel ); the Coomassie Blue-stained gel is shown in the lower panel. C , in vitro kinase assays were performed using purified GST-ASAP, GST-ASAP mutants (S289A or S369A/S370A), or GST as described in A and analyzed by SDS-PAGE and autoradiography ( upper panel ); the Coomassie Blue-stained gel is shown in the lower panel .

Techniques Used: In Vitro, Purification, Recombinant, Incubation, Mutagenesis, SDS Page, Autoradiography, Staining, Kinase Assay

58) Product Images from "c-Src-Mediated Phosphorylation of hnRNP K Drives Translational Activation of Specifically Silenced mRNAs"

Article Title: c-Src-Mediated Phosphorylation of hnRNP K Drives Translational Activation of Specifically Silenced mRNAs

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.13.4535-4543.2002

hnRNP K but not hnRNP E1 activates Src catalytic activity in vitro. Wild-type Src (wt) and the constitutively active Src(Y527F) mutant as a positive control were immunopurified from transfected human 293 HEK cells, and equal amounts were assayed with an excess of the substrate enolase in the presence of [γ- 32 P]ATP, incubated with increasing amounts (125 [+] and 375 [++] ng) of purified hnRNP K (lanes 3 and 4) or hnRNP E1 (lanes 7 and 8). The Src inhibitor PP2 (lanes 5 and 9) and the control PP3 (lanes 6 and 10) were added at a concentration of 1 μM. Phosphorylation of enolase and hnRNP K was analyzed by autoradiography (an example is shown in panel B). (A) The bars show the sum of the hnRNP K and enolase phosphorylation signals with the standard deviation observed in three repeat experiments.
Figure Legend Snippet: hnRNP K but not hnRNP E1 activates Src catalytic activity in vitro. Wild-type Src (wt) and the constitutively active Src(Y527F) mutant as a positive control were immunopurified from transfected human 293 HEK cells, and equal amounts were assayed with an excess of the substrate enolase in the presence of [γ- 32 P]ATP, incubated with increasing amounts (125 [+] and 375 [++] ng) of purified hnRNP K (lanes 3 and 4) or hnRNP E1 (lanes 7 and 8). The Src inhibitor PP2 (lanes 5 and 9) and the control PP3 (lanes 6 and 10) were added at a concentration of 1 μM. Phosphorylation of enolase and hnRNP K was analyzed by autoradiography (an example is shown in panel B). (A) The bars show the sum of the hnRNP K and enolase phosphorylation signals with the standard deviation observed in three repeat experiments.

Techniques Used: Activity Assay, In Vitro, Mutagenesis, Positive Control, Transfection, Incubation, Purification, Concentration Assay, Autoradiography, Standard Deviation

59) Product Images from "Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2"

Article Title: Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2

Journal: Journal of Virology

doi:

Full-length WT IEP86, but not mutant IEP86, is phosphorylated in a serum-inducible manner in vitro. Histidine-tagged full-length IEP86 purified from bacterial extracts was incubated with [γ- 32 P]ATP plus extract buffer alone (B) or with WCE prepared from U-373 MG cells which had been either serum starved (−) or serum stimulated (+) as described in Materials and Methods. His-tagged IEP86 was repurified and subjected to SDS-PAGE. Relative phosphorylation was detected by autoradiography. Mut. IEP86 represents full-length His-tagged IEP86 containing alanine substitution mutations at Thr 27 and Thr 233 /Ser 234 .
Figure Legend Snippet: Full-length WT IEP86, but not mutant IEP86, is phosphorylated in a serum-inducible manner in vitro. Histidine-tagged full-length IEP86 purified from bacterial extracts was incubated with [γ- 32 P]ATP plus extract buffer alone (B) or with WCE prepared from U-373 MG cells which had been either serum starved (−) or serum stimulated (+) as described in Materials and Methods. His-tagged IEP86 was repurified and subjected to SDS-PAGE. Relative phosphorylation was detected by autoradiography. Mut. IEP86 represents full-length His-tagged IEP86 containing alanine substitution mutations at Thr 27 and Thr 233 /Ser 234 .

Techniques Used: Mutagenesis, In Vitro, Purification, Incubation, SDS Page, Autoradiography

60) Product Images from "Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis"

Article Title: Caspase-Dependent Cleavage of c-Abl Contributes to Apoptosis

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.8.2790-2799.2003

c-Abl cleavage by caspase and c-Abl kinase activity are required to sensitize cells to apoptosis. (A) HuT78 cells were transfected with vector pcDNA3, with Abl-wt, or with Abl caspase-resistant mutant form Abl-TM, which carries all of the identified cleavage sites carrying the Asp-Ala mutation (D565A, D644A, and D958A), in the presence of a plasmid encoding GFP. At 24 h after transfection, cells were stimulated with 150 ng of anti-Fas antibody per ml. Transfected apoptotic cells were counted at different times, and specific apoptosis was calculated as described in Materials and Methods. The results shown are percentages of specific apoptosis at different times of stimulation. (B) HuT78 cells transfected with pcDNA3, Abl-wt, or Abl-Kin − and Fas stimulated as described for panel A. Specific apoptosis at different times of treatment was determined as described for panel A. (C) Abl constructs were transiently transfected into HuT78 cells. At 24 h after transfection, total protein extracts were prepared, separated by SDS-7.5% PAGE, blotted onto nitrocellulose, and probed with anti-Abl antibody K-12. ut, untransfected. WB, Western blot. (D) Endogenous c-Abl was extracted and immunoprecipitated with anti-Abl antibody K-12 from HuT78 cells after Fas treatment for different lengths of time. The activity of immunoprecipitated Abl was measured by in vitro kinase assay with the GST-c-Crk protein as the substrate in the presence of [γ- 32 P]ATP. Bands corresponding to the GST-c-Crk protein were excised, and incorporated radioactivity was measured by scintillation counting. The endogenous Abl activity after Fas stimulation for different lengths of time was normalized for the amount of immunoprecipitated Abl and is shown as fold activity.
Figure Legend Snippet: c-Abl cleavage by caspase and c-Abl kinase activity are required to sensitize cells to apoptosis. (A) HuT78 cells were transfected with vector pcDNA3, with Abl-wt, or with Abl caspase-resistant mutant form Abl-TM, which carries all of the identified cleavage sites carrying the Asp-Ala mutation (D565A, D644A, and D958A), in the presence of a plasmid encoding GFP. At 24 h after transfection, cells were stimulated with 150 ng of anti-Fas antibody per ml. Transfected apoptotic cells were counted at different times, and specific apoptosis was calculated as described in Materials and Methods. The results shown are percentages of specific apoptosis at different times of stimulation. (B) HuT78 cells transfected with pcDNA3, Abl-wt, or Abl-Kin − and Fas stimulated as described for panel A. Specific apoptosis at different times of treatment was determined as described for panel A. (C) Abl constructs were transiently transfected into HuT78 cells. At 24 h after transfection, total protein extracts were prepared, separated by SDS-7.5% PAGE, blotted onto nitrocellulose, and probed with anti-Abl antibody K-12. ut, untransfected. WB, Western blot. (D) Endogenous c-Abl was extracted and immunoprecipitated with anti-Abl antibody K-12 from HuT78 cells after Fas treatment for different lengths of time. The activity of immunoprecipitated Abl was measured by in vitro kinase assay with the GST-c-Crk protein as the substrate in the presence of [γ- 32 P]ATP. Bands corresponding to the GST-c-Crk protein were excised, and incorporated radioactivity was measured by scintillation counting. The endogenous Abl activity after Fas stimulation for different lengths of time was normalized for the amount of immunoprecipitated Abl and is shown as fold activity.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Construct, Polyacrylamide Gel Electrophoresis, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Radioactivity

61) Product Images from "Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase"

Article Title: Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

Journal: Journal of Virology

doi:

Sensitivity of core particle-associated kinase to different kinase inhibitors. The activity of the core particle-associated kinase from intracellular core particles either without the proteinase K pretreatment (A) or with the proteinase K (25 ng) pretreatment (B) was assayed by the incorporation of [γ- 32 P]ATP into core protein. The kinase assay was performed in the absence (lane 1) or presence of kinase inhibitors: 25 U of heparin per ml (lane 2), 500 mM spermidine (lane 3), 5 mM manganese (lane 4), 6 mM DRB (lane 5), 100 mM H89 (lane 6), and 100 mM H7 (lane 7).
Figure Legend Snippet: Sensitivity of core particle-associated kinase to different kinase inhibitors. The activity of the core particle-associated kinase from intracellular core particles either without the proteinase K pretreatment (A) or with the proteinase K (25 ng) pretreatment (B) was assayed by the incorporation of [γ- 32 P]ATP into core protein. The kinase assay was performed in the absence (lane 1) or presence of kinase inhibitors: 25 U of heparin per ml (lane 2), 500 mM spermidine (lane 3), 5 mM manganese (lane 4), 6 mM DRB (lane 5), 100 mM H89 (lane 6), and 100 mM H7 (lane 7).

Techniques Used: Activity Assay, Kinase Assay

Detection of the CAK by an in-gel kinase assay. Proteins (20 μg) from 0.4 M KCl (lane 1) and 0.2 M KCl (lane 2) eluted fractions were separated by SDS-PAGE (12% polyacrylamide) with GST-2/5C protein as the substrate. After denaturation and renaturation, proteins in the gel were incubated with [γ- 32 P]ATP and tested for kinase activity. After being washed, the protein(s) containing the kinase activity was visualized by autoradiography.
Figure Legend Snippet: Detection of the CAK by an in-gel kinase assay. Proteins (20 μg) from 0.4 M KCl (lane 1) and 0.2 M KCl (lane 2) eluted fractions were separated by SDS-PAGE (12% polyacrylamide) with GST-2/5C protein as the substrate. After denaturation and renaturation, proteins in the gel were incubated with [γ- 32 P]ATP and tested for kinase activity. After being washed, the protein(s) containing the kinase activity was visualized by autoradiography.

Techniques Used: Kinase Assay, SDS Page, Incubation, Activity Assay, Autoradiography

62) Product Images from "Decreased Cell Wall Digestibility in Canola Transformed with Chimeric Tyrosine Decarboxylase Genes from Opium Poppy 1"

Article Title: Decreased Cell Wall Digestibility in Canola Transformed with Chimeric Tyrosine Decarboxylase Genes from Opium Poppy 1

Journal: Plant Physiology

doi:

NPT II dot-blot assay of leaf extracts from putative T 0 transgenic canola plants. Labeling reactions were performed with [γ- 32 P]ATP and plant extracts in the presence (+Kan) or absence (−Kan) of kanamycin. The negative control was wild-type (WT) canola. BN10 and BN11 were transformed with 35S::TYDC1 , whereas all other plants were transformed with 35S::TYDC2 .
Figure Legend Snippet: NPT II dot-blot assay of leaf extracts from putative T 0 transgenic canola plants. Labeling reactions were performed with [γ- 32 P]ATP and plant extracts in the presence (+Kan) or absence (−Kan) of kanamycin. The negative control was wild-type (WT) canola. BN10 and BN11 were transformed with 35S::TYDC1 , whereas all other plants were transformed with 35S::TYDC2 .

Techniques Used: Dot Blot, Transgenic Assay, Labeling, Negative Control, Transformation Assay

63) Product Images from "Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen"

Article Title: Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0308164100

Photoaffinity labeling of thylakoids (T) or PSII core complexes (PSII) with 25 μM [α- 32 P]8-N 3 GTP ( A ) and 5 μM [γ- 32 P]8-N 3 ATP ( B ). Samples diluted to 0.3 mg/ml Chl in buffer A were first dark-incubated (–) or preilluminated (+) on ice for 30 min, followed by incubation with the photoprobe for 3 min in darkness. The samples were photolysed with UV-C light for 90 s on ice and subjected to SDS/PAGE and phosphorimaging. ( C ) Identification of the GTP-binding protein as the OEC33. PSII core complexes were photolabeled with 50μM [α- 32 P]8-N 3 GTP as described above followed by alkaline Tris-washing, centrifugation, and separation of proteins by SDS/PAGE in the pellet (Pel.) and the supernatant (Sup.). The phosphorimage in the 33-kDa region and the corresponding Western blot with anti-OEC33 antibody are shown.
Figure Legend Snippet: Photoaffinity labeling of thylakoids (T) or PSII core complexes (PSII) with 25 μM [α- 32 P]8-N 3 GTP ( A ) and 5 μM [γ- 32 P]8-N 3 ATP ( B ). Samples diluted to 0.3 mg/ml Chl in buffer A were first dark-incubated (–) or preilluminated (+) on ice for 30 min, followed by incubation with the photoprobe for 3 min in darkness. The samples were photolysed with UV-C light for 90 s on ice and subjected to SDS/PAGE and phosphorimaging. ( C ) Identification of the GTP-binding protein as the OEC33. PSII core complexes were photolabeled with 50μM [α- 32 P]8-N 3 GTP as described above followed by alkaline Tris-washing, centrifugation, and separation of proteins by SDS/PAGE in the pellet (Pel.) and the supernatant (Sup.). The phosphorimage in the 33-kDa region and the corresponding Western blot with anti-OEC33 antibody are shown.

Techniques Used: Labeling, Incubation, SDS Page, Binding Assay, Centrifugation, Western Blot

Photolabeling of the OEC33 and the 36.5-kDa proteins. ( A ) PSII core complexes were preilluminated and photolysed with the indicated concentrations of [α- 32 P]8-N 3 GTP followed by SDS/PAGE and phosphorimaging ( Inset ). The 32 P incorporation into the OEC33 band was quantified, and the maximal value was set as 100%. ( B ) The photolabeling of the OEC33 protein with 25 μM [α- 32 P]8-N 3 GTP was carried out in preilluminated PSII core complexes in the presence of 250 μM nonlabeled nucleotides. The photolabeling of the 36.5-kDa protein with 7 μM [γ- 32 P]8-N 3 ATP was carried out in preilluminated thylakoids in the presence of 200 μM nonlabeled nucleotides. Values represent the percentage of remaining photolabeling compared with the value obtained in the absence of nonlabeled nucleotides (n.d., not determined). ( C ) Phosphorimage of SDS/PAGE containing isolated OEC33 protein photolabeled with 10 μM[α- 32 P]GTP in the presence of the indicated concentrations of nonlabeled GTP.
Figure Legend Snippet: Photolabeling of the OEC33 and the 36.5-kDa proteins. ( A ) PSII core complexes were preilluminated and photolysed with the indicated concentrations of [α- 32 P]8-N 3 GTP followed by SDS/PAGE and phosphorimaging ( Inset ). The 32 P incorporation into the OEC33 band was quantified, and the maximal value was set as 100%. ( B ) The photolabeling of the OEC33 protein with 25 μM [α- 32 P]8-N 3 GTP was carried out in preilluminated PSII core complexes in the presence of 250 μM nonlabeled nucleotides. The photolabeling of the 36.5-kDa protein with 7 μM [γ- 32 P]8-N 3 ATP was carried out in preilluminated thylakoids in the presence of 200 μM nonlabeled nucleotides. Values represent the percentage of remaining photolabeling compared with the value obtained in the absence of nonlabeled nucleotides (n.d., not determined). ( C ) Phosphorimage of SDS/PAGE containing isolated OEC33 protein photolabeled with 10 μM[α- 32 P]GTP in the presence of the indicated concentrations of nonlabeled GTP.

Techniques Used: SDS Page, Isolation

64) Product Images from "Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel 1"

Article Title: Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel 1

Journal: Plant Physiology

doi:

CDPK phosphorylates KAT1 protein in a Ca 2+ -dependent manner. The membrane (Mem; A, lanes 1–6, and 10) or supernatant (Sup; B, lanes 1–3) fractions of the product translated in the presence of microsome membranes were subjected to phosphorylation by CDPK in the presence of [γ- 32 P]ATP and Ca 2+ . A, Lane 8, and B, lane 5, [ 35 S]Met-labeled translation product from the KAT1 cDNA template. A, Lane 7 and B, lane 4, [ 35 S]Met-labeled translation product without DNA template. In lane 9, the microsome membranes were added to the translation system just after translation and then phosphorylated by CDPK in the presence of Ca 2+ . The arrows indicate the position of the 35 S-labeled KAT1 protein; note the corresponding phosphorylated band in lanes 6 and 10. The protein samples (20 μg of protein per lane) were resolved on 9% polyacrylamide gels. −, Absent; +, present.
Figure Legend Snippet: CDPK phosphorylates KAT1 protein in a Ca 2+ -dependent manner. The membrane (Mem; A, lanes 1–6, and 10) or supernatant (Sup; B, lanes 1–3) fractions of the product translated in the presence of microsome membranes were subjected to phosphorylation by CDPK in the presence of [γ- 32 P]ATP and Ca 2+ . A, Lane 8, and B, lane 5, [ 35 S]Met-labeled translation product from the KAT1 cDNA template. A, Lane 7 and B, lane 4, [ 35 S]Met-labeled translation product without DNA template. In lane 9, the microsome membranes were added to the translation system just after translation and then phosphorylated by CDPK in the presence of Ca 2+ . The arrows indicate the position of the 35 S-labeled KAT1 protein; note the corresponding phosphorylated band in lanes 6 and 10. The protein samples (20 μg of protein per lane) were resolved on 9% polyacrylamide gels. −, Absent; +, present.

Techniques Used: Labeling

The 57-kD kinase exhibits a Ca 2+ -dependent electrophoretic mobility shift in both autophosphorylation and in-gel activity assays. Ca 2+ or EGTA to a final concentration of 2 mm was added to the GCP-soluble proteins dissolved in SDS-PAGE sample buffer. The Ca 2+ - and EGTA-treated samples were loaded (A and B, 40 μg of protein per lane; C and D, 20 μg of protein per lane) with a blank lane between the two samples and resolved on 12% polyacrylamide gels. Autophosphorylation (A and B) and in-gel kinase activity (C and D) assays were performed by incubating the renatured gels with [γ- 32 P]ATP in the presence of 100 μm free Ca 2+ (A and C) or 450 μm EGTA (B and D). The presence of histone III-S (0.5 mg mL −1 ) in the polyacrylamide separating gel precludes protein staining; therefore, prestained protein molecular mass standards are used in the kinase activity assay (C and D). The arrows indicate the positions of the 57-kD kinase with a Ca 2+ -induced electrophoretic mobility shift. The asterisks indicate the positions of the 38-kD kinase that does not exhibit a Ca 2+ -induced electrophoretic mobility shift.
Figure Legend Snippet: The 57-kD kinase exhibits a Ca 2+ -dependent electrophoretic mobility shift in both autophosphorylation and in-gel activity assays. Ca 2+ or EGTA to a final concentration of 2 mm was added to the GCP-soluble proteins dissolved in SDS-PAGE sample buffer. The Ca 2+ - and EGTA-treated samples were loaded (A and B, 40 μg of protein per lane; C and D, 20 μg of protein per lane) with a blank lane between the two samples and resolved on 12% polyacrylamide gels. Autophosphorylation (A and B) and in-gel kinase activity (C and D) assays were performed by incubating the renatured gels with [γ- 32 P]ATP in the presence of 100 μm free Ca 2+ (A and C) or 450 μm EGTA (B and D). The presence of histone III-S (0.5 mg mL −1 ) in the polyacrylamide separating gel precludes protein staining; therefore, prestained protein molecular mass standards are used in the kinase activity assay (C and D). The arrows indicate the positions of the 57-kD kinase with a Ca 2+ -induced electrophoretic mobility shift. The asterisks indicate the positions of the 38-kD kinase that does not exhibit a Ca 2+ -induced electrophoretic mobility shift.

Techniques Used: Electrophoretic Mobility Shift Assay, Activity Assay, Concentration Assay, SDS Page, Staining, Kinase Assay

Ca 2+ stimulates phosphorylation of certain guard cell proteins. Protein phosphorylation was performed by incubating 30 μg of soluble (S) proteins (A) or membrane (M) proteins (B) with [γ- 32 P]ATP in the presence of various concentrations of free Ca 2+ ([Ca 2+ ] f ). Phosphorylation of soluble proteins (30 μg) in the presence of 10 μm CsA (lanes +) or 0.2% ethanol (lanes −) at nominally 0 or 1 μm free Ca 2+ . The phosphoproteins were resolved on 5 to 20% gradient polyacrylamide gels. The arrows indicate the positions of proteins with Ca 2+ -stimulated phosphorylation. The asterisk indicates the position of a protein with CsA-enhanced phosphorylation. The 120- and 52-kD proteins that exhibited Ca 2+ -stimulated phosphorylation (indicated by the highest and the lowest arrows) also exhibited CsA-enhanced phosphorylation.
Figure Legend Snippet: Ca 2+ stimulates phosphorylation of certain guard cell proteins. Protein phosphorylation was performed by incubating 30 μg of soluble (S) proteins (A) or membrane (M) proteins (B) with [γ- 32 P]ATP in the presence of various concentrations of free Ca 2+ ([Ca 2+ ] f ). Phosphorylation of soluble proteins (30 μg) in the presence of 10 μm CsA (lanes +) or 0.2% ethanol (lanes −) at nominally 0 or 1 μm free Ca 2+ . The phosphoproteins were resolved on 5 to 20% gradient polyacrylamide gels. The arrows indicate the positions of proteins with Ca 2+ -stimulated phosphorylation. The asterisk indicates the position of a protein with CsA-enhanced phosphorylation. The 120- and 52-kD proteins that exhibited Ca 2+ -stimulated phosphorylation (indicated by the highest and the lowest arrows) also exhibited CsA-enhanced phosphorylation.

Techniques Used:

65) Product Images from "Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain"

Article Title: Autoinhibition of Jak2 Tyrosine Kinase Is Dependent on Specific Regions in Its Pseudokinase Domain

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-06-0342

Comparison of the effect of two different JH2 deletions on the activity of Jak2. (A) Schematic presentation of Jak2 constructs. (B) Expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA were transfected into 293T cells and cell lysates were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates were separated in 7.5% SDS-PAGE and analyzed in anti-phosphotyrosine (top) and anti-HA immunoblots (middle). Aliquots of cell lysates were separated in 7.5% SDS-PAGE and analyzed in anti-HA immunoblot (bottom). (C) Expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA were transfected into 293T cells, and cell lysates were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates were subjected to in vitro kinase assay by using [γ- 32 P]ATP and Stat5-derived peptide as a substrate. The peptides were separated in 20% SDS-PAGE followed by quantification using PhosphorImager. Aliquots of the immunoprecipitates (top) and cell lysates (bottom) were also separated in 7.5% SDS-PAGE and analyzed in anti-HA immunoblot. (D) Stat5 expression plasmid was transfected either alone or together with expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA into 293T cells, and cell lysates were immunoprecipitated using anti-Stat5 antibody. Aliquots of the immunoprecipitates were separated in 7.5% SDS-PAGE and analyzed in anti-phosphotyrosine (top) and anti-Stat5 (middle) immunoblots. Cell lysates were separated in 7.5% SDS-PAGE and analyzed by immunoblotting with anti-HA antibody (bottom). The mobilities of the molecular mass markers (in kilodaltons) are shown on the right.
Figure Legend Snippet: Comparison of the effect of two different JH2 deletions on the activity of Jak2. (A) Schematic presentation of Jak2 constructs. (B) Expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA were transfected into 293T cells and cell lysates were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates were separated in 7.5% SDS-PAGE and analyzed in anti-phosphotyrosine (top) and anti-HA immunoblots (middle). Aliquots of cell lysates were separated in 7.5% SDS-PAGE and analyzed in anti-HA immunoblot (bottom). (C) Expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA were transfected into 293T cells, and cell lysates were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates were subjected to in vitro kinase assay by using [γ- 32 P]ATP and Stat5-derived peptide as a substrate. The peptides were separated in 20% SDS-PAGE followed by quantification using PhosphorImager. Aliquots of the immunoprecipitates (top) and cell lysates (bottom) were also separated in 7.5% SDS-PAGE and analyzed in anti-HA immunoblot. (D) Stat5 expression plasmid was transfected either alone or together with expression plasmids for Jak2-HA, JH2Δ-HA, and Bgl IIΔ-HA into 293T cells, and cell lysates were immunoprecipitated using anti-Stat5 antibody. Aliquots of the immunoprecipitates were separated in 7.5% SDS-PAGE and analyzed in anti-phosphotyrosine (top) and anti-Stat5 (middle) immunoblots. Cell lysates were separated in 7.5% SDS-PAGE and analyzed by immunoblotting with anti-HA antibody (bottom). The mobilities of the molecular mass markers (in kilodaltons) are shown on the right.

Techniques Used: Activity Assay, Construct, Expressing, Transfection, Immunoprecipitation, SDS Page, Western Blot, In Vitro, Kinase Assay, Derivative Assay, Plasmid Preparation

66) Product Images from "Bacillus subtilis RecN binds and protects 3?-single-stranded DNA extensions in the presence of ATP"

Article Title: Bacillus subtilis RecN binds and protects 3?-single-stranded DNA extensions in the presence of ATP

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki533

DNA binding by RecN. ( A ) [ 32 P]Labelled ssDNA 60 (1 nM), at the 5′-terminus, was incubated with RecN (0.4, 0.8, 1.7, 3.7, 15, 30 and 60 nM) for 30 min at 37°C in buffer B in the absence (lanes 1–9) or presence of 1 mM ADP (lanes 10–18) or 1 mM ATP (lanes 19–27). RecN was not added to the controls in lane 1, 10 and 19. In lanes 10 and 19, (+) denotes that identical results were observed in the presence or absence of a nucleotide cofactor. ( B ) [γ- 32 P]Labelled ssDNA 60 (1 nM) and RecN (10 nM) were incubated and then ADP (0.01, 0.05, 0.1, 0.5 and 1 mM, lanes 3–7), ATP (0.01–1 mM, lanes 8–12) or AMP-PNP (0.01–1 mM, lanes 13–16) was added; incubation continued for 30 min at 37°C in buffer B. RecN was not added in lane 1. Complexes formed were separated by 10% ndPAGE and visualized by autoradiography. FD, free DNA; CI, complex I; CII, complex II; CIII, complex III; + and −, the presence or absence of the indicated factor.
Figure Legend Snippet: DNA binding by RecN. ( A ) [ 32 P]Labelled ssDNA 60 (1 nM), at the 5′-terminus, was incubated with RecN (0.4, 0.8, 1.7, 3.7, 15, 30 and 60 nM) for 30 min at 37°C in buffer B in the absence (lanes 1–9) or presence of 1 mM ADP (lanes 10–18) or 1 mM ATP (lanes 19–27). RecN was not added to the controls in lane 1, 10 and 19. In lanes 10 and 19, (+) denotes that identical results were observed in the presence or absence of a nucleotide cofactor. ( B ) [γ- 32 P]Labelled ssDNA 60 (1 nM) and RecN (10 nM) were incubated and then ADP (0.01, 0.05, 0.1, 0.5 and 1 mM, lanes 3–7), ATP (0.01–1 mM, lanes 8–12) or AMP-PNP (0.01–1 mM, lanes 13–16) was added; incubation continued for 30 min at 37°C in buffer B. RecN was not added in lane 1. Complexes formed were separated by 10% ndPAGE and visualized by autoradiography. FD, free DNA; CI, complex I; CII, complex II; CIII, complex III; + and −, the presence or absence of the indicated factor.

Techniques Used: Binding Assay, Incubation, Autoradiography

67) Product Images from "p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2C?"

Article Title: p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2C?

Journal: Biochemical Journal

doi: 10.1042/BJ20040948

Phosphorylation of PP2Cδ ( A ) HEK-293 cells were serum-starved for 4 h and incubated in the absence or presence of 50 nM PMA for 20 min. PMA-treated cells were preincubated with 10 μM U0126 for 40 min. The cells were lysed, subjected to SDS/PAGE and immunoblotted with anti-PP2Cδ antibody or anti-(active MAPK) antibody. ( B ) COS-7 cells were transfected with an empty vector or a plasmid expressing GST–PP2Cδ. Wild-type PP2Cδ, mutant PP2Cδ(T315A), PP2Cδ(T333A) and PP2Cδ(T315A,T333A) were incubated with activated ERK in the presence of [γ- 32 P]ATP and analysed by SDS/PAGE and autoradiography. ( C ) Four members of the PP2C family were aligned using the Vector NTI. Identical residues are shaded. The two putative ERK phosphorylation sites in PP2Cδ are shown in bold.
Figure Legend Snippet: Phosphorylation of PP2Cδ ( A ) HEK-293 cells were serum-starved for 4 h and incubated in the absence or presence of 50 nM PMA for 20 min. PMA-treated cells were preincubated with 10 μM U0126 for 40 min. The cells were lysed, subjected to SDS/PAGE and immunoblotted with anti-PP2Cδ antibody or anti-(active MAPK) antibody. ( B ) COS-7 cells were transfected with an empty vector or a plasmid expressing GST–PP2Cδ. Wild-type PP2Cδ, mutant PP2Cδ(T315A), PP2Cδ(T333A) and PP2Cδ(T315A,T333A) were incubated with activated ERK in the presence of [γ- 32 P]ATP and analysed by SDS/PAGE and autoradiography. ( C ) Four members of the PP2C family were aligned using the Vector NTI. Identical residues are shaded. The two putative ERK phosphorylation sites in PP2Cδ are shown in bold.

Techniques Used: Incubation, SDS Page, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Autoradiography

68) Product Images from "Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens"

Article Title: Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens

Journal: Chemistry & biology

doi: 10.1016/j.chembiol.2009.10.014

TLC analysis of the product generated by Rv1011 I. a. The reaction mixtures containing CDP-ME, MgCl 2 , and [γ- 32 P]ATP without Rv1011 I (lane 1) and with Rv1011 I (lane 2). b. Co-migration experiment with a reaction containing [γ- 32 P]ATP,
Figure Legend Snippet: TLC analysis of the product generated by Rv1011 I. a. The reaction mixtures containing CDP-ME, MgCl 2 , and [γ- 32 P]ATP without Rv1011 I (lane 1) and with Rv1011 I (lane 2). b. Co-migration experiment with a reaction containing [γ- 32 P]ATP,

Techniques Used: Thin Layer Chromatography, Generated, Migration

69) Product Images from "A Novel Mammalian, Mitotic Spindle-associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators "

Article Title: A Novel Mammalian, Mitotic Spindle-associated Kinase Is Related to Yeast and Fly Chromosome Segregation Regulators

Journal: The Journal of Cell Biology

doi:

The mutant IAK1 D287N shows reduced kinase activity. Cell extracts from NIH 3T3 cells transiently expressing the flag-tagged IAK1 and IAK1 D287N constructs were prepared, and the wild-type and the mutant form of the kinase was selectively isolated using the anti–flag M2 affinity gel as described in the Materials and Methods section. Mock-transfected cells were used as the control. The affinity-purified wild-type and mutant form of the IAK1 kinase was used for the in vitro kinase activity using myelin basic protein as the substrate. The experiment was repeated three times, and the typical result from a single experiment is presented here. The incorporation of [γ- 32 P]ATP into myelin basic protein was measured using a Molecular Dynamics phosphoimager, and the result is presented in the form of a bar diagram after background correction. The inset shows the autoradiographic representation of the same result. Lane 1 , control mock-transfected cells; lane 2 , IAK1; lane 3 , IAK1 D287N .
Figure Legend Snippet: The mutant IAK1 D287N shows reduced kinase activity. Cell extracts from NIH 3T3 cells transiently expressing the flag-tagged IAK1 and IAK1 D287N constructs were prepared, and the wild-type and the mutant form of the kinase was selectively isolated using the anti–flag M2 affinity gel as described in the Materials and Methods section. Mock-transfected cells were used as the control. The affinity-purified wild-type and mutant form of the IAK1 kinase was used for the in vitro kinase activity using myelin basic protein as the substrate. The experiment was repeated three times, and the typical result from a single experiment is presented here. The incorporation of [γ- 32 P]ATP into myelin basic protein was measured using a Molecular Dynamics phosphoimager, and the result is presented in the form of a bar diagram after background correction. The inset shows the autoradiographic representation of the same result. Lane 1 , control mock-transfected cells; lane 2 , IAK1; lane 3 , IAK1 D287N .

Techniques Used: Mutagenesis, Activity Assay, Expressing, Construct, Isolation, Transfection, Affinity Purification, In Vitro

70) Product Images from "Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases"

Article Title: Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg298

Fig. 8. Functional relationship of Gad8p with Ksg1p. ( A ) Suppression of ksg1-208 by overexpression of gad8 . The ksg1-208 mutant (JW237) was transformed with pREP41- gad8 , pREP41- gad8-T387A , pREP41- gad8-S546A , pREP41- gad8-S546D , pREP41- ksg1 and the vector pREP41. Each transformant was examined for mating efficiency (left panel) and growth at the restrictive temperature (right panel). Transformants were cultured on SSA at 26.5°C for six days before scoring mating efficiency, and on MM medium at 33°C for three days to estimate temperature sensitivity. ( B ) The ksg1-208 mutation affects the kinase activity of Gad8p. Cells expressing Gad8-6HAp in either the wild-type (JW960) or the ksg1-208 (JW967) background were grown to logarithmic phase in YE liquid medium at 26.5°C and shifted to 34°C. Cells were harvested before and 4.5 h after the shift, and Gad8-6HAp was immunopurified from each sample. The kinase activity of Gad8-6HAp from each sample was assayed in vitro using crosstide. Immunoprecipitated Gad8-6HAp, detected by western blotting, is also shown. ( C ) Ksg1p phosphorylates Gad8p in vitro . Wild-type and mutant Gad8p fused to MBP, expressed bacterially and immunopurified, was incubated with GST-tagged Ksg1p in the presence of [γ- 32 P]ATP. Phosphorylation of MBP–Gad8p was detected by autoradiography. Ksg1-K128Rp, a kinase-dead form of Ksg1, was also examined as a negative control. An autoradiograph is shown in the upper panel and a Coomassie staining pattern in shown in the lower panel. ( D ) Inhibition of the Ksg1p kinase activity by PIF-tide. Phosphorylation reaction of Gad8p and Gad8-S546Dp was conducted as described in (C), in the presence of PIF-tide at the concentration indicated.
Figure Legend Snippet: Fig. 8. Functional relationship of Gad8p with Ksg1p. ( A ) Suppression of ksg1-208 by overexpression of gad8 . The ksg1-208 mutant (JW237) was transformed with pREP41- gad8 , pREP41- gad8-T387A , pREP41- gad8-S546A , pREP41- gad8-S546D , pREP41- ksg1 and the vector pREP41. Each transformant was examined for mating efficiency (left panel) and growth at the restrictive temperature (right panel). Transformants were cultured on SSA at 26.5°C for six days before scoring mating efficiency, and on MM medium at 33°C for three days to estimate temperature sensitivity. ( B ) The ksg1-208 mutation affects the kinase activity of Gad8p. Cells expressing Gad8-6HAp in either the wild-type (JW960) or the ksg1-208 (JW967) background were grown to logarithmic phase in YE liquid medium at 26.5°C and shifted to 34°C. Cells were harvested before and 4.5 h after the shift, and Gad8-6HAp was immunopurified from each sample. The kinase activity of Gad8-6HAp from each sample was assayed in vitro using crosstide. Immunoprecipitated Gad8-6HAp, detected by western blotting, is also shown. ( C ) Ksg1p phosphorylates Gad8p in vitro . Wild-type and mutant Gad8p fused to MBP, expressed bacterially and immunopurified, was incubated with GST-tagged Ksg1p in the presence of [γ- 32 P]ATP. Phosphorylation of MBP–Gad8p was detected by autoradiography. Ksg1-K128Rp, a kinase-dead form of Ksg1, was also examined as a negative control. An autoradiograph is shown in the upper panel and a Coomassie staining pattern in shown in the lower panel. ( D ) Inhibition of the Ksg1p kinase activity by PIF-tide. Phosphorylation reaction of Gad8p and Gad8-S546Dp was conducted as described in (C), in the presence of PIF-tide at the concentration indicated.

Techniques Used: Functional Assay, Over Expression, Mutagenesis, Transformation Assay, Plasmid Preparation, Cell Culture, Activity Assay, Expressing, In Vitro, Immunoprecipitation, Western Blot, Incubation, Autoradiography, Negative Control, Staining, Inhibition, Concentration Assay

71) Product Images from "Phosphorylated hMSH6: DNA mismatch versus DNA damage recognition"

Article Title: Phosphorylated hMSH6: DNA mismatch versus DNA damage recognition

Journal: Mutation research

doi: 10.1016/j.mrfmmm.2010.10.008

Pretreatment with TPA and/or G:T mismatch increases hMSH6 phosphorylation, pretreatment with UCN-01 decreases hMSH6 phosphorylation A . HeLa MR cells were untreated or treated with 200 nM TPA for 12 h, and nuclear extracts were incubated with or without 200 ng nonradioactive G:C or G:T oligomer and 50 µCi of [γ- 32 P]-ATP for 30 min. hMSH6 was immunoprecipitated, subjected to SDS PAGE, dried and subjected to autoradiography, with a typical autoradiograph shown. The MSH6 immunoblot indicates comparable amounts of hMSH6 protein in each preparation. The bar graph depicts relative ratios of each condition (untreated controls normalized to a value of 1) from three experiments subjected to phosphorimager quantification of hMSH6 radioactive bands. Asterick (*) indicate statistically significant difference from untreated control (Student t-test, P
Figure Legend Snippet: Pretreatment with TPA and/or G:T mismatch increases hMSH6 phosphorylation, pretreatment with UCN-01 decreases hMSH6 phosphorylation A . HeLa MR cells were untreated or treated with 200 nM TPA for 12 h, and nuclear extracts were incubated with or without 200 ng nonradioactive G:C or G:T oligomer and 50 µCi of [γ- 32 P]-ATP for 30 min. hMSH6 was immunoprecipitated, subjected to SDS PAGE, dried and subjected to autoradiography, with a typical autoradiograph shown. The MSH6 immunoblot indicates comparable amounts of hMSH6 protein in each preparation. The bar graph depicts relative ratios of each condition (untreated controls normalized to a value of 1) from three experiments subjected to phosphorimager quantification of hMSH6 radioactive bands. Asterick (*) indicate statistically significant difference from untreated control (Student t-test, P

Techniques Used: Incubation, Immunoprecipitation, SDS Page, Autoradiography

72) Product Images from "High CO2 Levels Impair Alveolar Epithelial Function Independently of pH"

Article Title: High CO2 Levels Impair Alveolar Epithelial Function Independently of pH

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001238

Na,K-ATPase function is impaired in rat lungs exposed to hypercapnic acidosis. (A) Basolateral membranes (BLM) were purified from the peripheral lung tissue of rat lungs exposed to 40 mmHg CO 2 (pH e : 7.4) or 60 mmHg CO 2 (pH e : 7.2), and Na,K-ATPase activity was measured as [γ- 32 P]ATP hydrolysis. Graph represents the mean±SEM, (n = 3). (B) BLM and total membranes were purified from the peripheral lung tissue of rat lungs treated as (A), and Na,K-ATPase protein abundance was assessed by Western blot. Graph represents the mean±SEM, (n = 3). Representative blots of Na,K-ATPase α 1 -subunit at the BLM and total membrane protein abundance are shown. pH e : extracellular pH. * p
Figure Legend Snippet: Na,K-ATPase function is impaired in rat lungs exposed to hypercapnic acidosis. (A) Basolateral membranes (BLM) were purified from the peripheral lung tissue of rat lungs exposed to 40 mmHg CO 2 (pH e : 7.4) or 60 mmHg CO 2 (pH e : 7.2), and Na,K-ATPase activity was measured as [γ- 32 P]ATP hydrolysis. Graph represents the mean±SEM, (n = 3). (B) BLM and total membranes were purified from the peripheral lung tissue of rat lungs treated as (A), and Na,K-ATPase protein abundance was assessed by Western blot. Graph represents the mean±SEM, (n = 3). Representative blots of Na,K-ATPase α 1 -subunit at the BLM and total membrane protein abundance are shown. pH e : extracellular pH. * p

Techniques Used: Purification, Activity Assay, Western Blot

High CO 2 levels impair Na, K-ATPase activity independently of pH. (A) ATII cells were exposed to 40, 60, 80 and 120 mmHg CO 2 with extracellular pH (pH e ): 7.4 or to 40 mmHg CO 2 with pH e : 7.2 for 30 min, and Na,K-ATPase activity was measured as [γ- 32 P]ATP hydrolysis. Graph represents the mean±SEM, (n = 5). (B) ATII cells were treated as described in (A) and the Na,K-ATPase protein abundance at the plasma membrane (PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α 1 -subunit at the PM and total protein abundance are shown. (C) Live cell imaging of GFPα 1 -A549. Cells were exposed to 40 mmHg CO 2 (pH e : 7.4) for 10 min (left panels) and then switched to 120 mmHg CO 2 (pH e : 7.4) (right, upper panel) or to 40 mmHg CO 2 (pH e : 7.2) (right, lower panel) for 30 min. White arrows indicate the plasma membrane Na,K-ATPase. (D) Live cell imaging of GFPα 1 -A549. Cells were exposed to 40 mmHg CO 2 (pH e : 7.4) for 10 min (left panel), switched to 120 mmHg CO 2 (pH e : 7.4) (middle panel) for 30 min and switched back to 40 mmHg CO 2 (pH e : 7.4) for 60 min (left panel). White arrows indicate the plasma membrane Na,K-ATPase * p
Figure Legend Snippet: High CO 2 levels impair Na, K-ATPase activity independently of pH. (A) ATII cells were exposed to 40, 60, 80 and 120 mmHg CO 2 with extracellular pH (pH e ): 7.4 or to 40 mmHg CO 2 with pH e : 7.2 for 30 min, and Na,K-ATPase activity was measured as [γ- 32 P]ATP hydrolysis. Graph represents the mean±SEM, (n = 5). (B) ATII cells were treated as described in (A) and the Na,K-ATPase protein abundance at the plasma membrane (PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α 1 -subunit at the PM and total protein abundance are shown. (C) Live cell imaging of GFPα 1 -A549. Cells were exposed to 40 mmHg CO 2 (pH e : 7.4) for 10 min (left panels) and then switched to 120 mmHg CO 2 (pH e : 7.4) (right, upper panel) or to 40 mmHg CO 2 (pH e : 7.2) (right, lower panel) for 30 min. White arrows indicate the plasma membrane Na,K-ATPase. (D) Live cell imaging of GFPα 1 -A549. Cells were exposed to 40 mmHg CO 2 (pH e : 7.4) for 10 min (left panel), switched to 120 mmHg CO 2 (pH e : 7.4) (middle panel) for 30 min and switched back to 40 mmHg CO 2 (pH e : 7.4) for 60 min (left panel). White arrows indicate the plasma membrane Na,K-ATPase * p

Techniques Used: Activity Assay, Western Blot, Live Cell Imaging

73) Product Images from "HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity"

Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm525

HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.
Figure Legend Snippet: HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.

Techniques Used: Inhibition, Activity Assay, Agarose Gel Electrophoresis, Electrophoresis, Staining, Plasmid Preparation, Thin Layer Chromatography

74) Product Images from "A Novel "Four-component" Two-component Signal Transduction Mechanism Regulates Developmental Progression in Myxococcus xanthus *"

Article Title: A Novel "Four-component" Two-component Signal Transduction Mechanism Regulates Developmental Progression in Myxococcus xanthus *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.033415

A , domain architecture of RedC–F two-component signaling homologs. Shown is a schematic diagram of conserved domains detected by SMART ( 40 , 41 ) in RedC histidine kinase, RedE histidine kinase-like protein, and RedD and RedF response regulator proteins. Gray rectangles , transmembrane segments predicted by TMpred Server (available on the World Wide Web); white squares , HisKA phosphoaccepting/dimerization domain; white triangles , HATPase_c ATP binding and hydrolysis catalytic domain; white circles , REC receiver domain. Amino acid positions of the invariant histidine ( H ) and aspartic acid ( D ) residues predicted to be the sites of phosphorylation are identified in the relevant domains. B–E , autophosphorylation analysis of Red TCS homologs. Shown are 10 μ m purified histidine kinase RedC-T ( B ) or histidine kinase-like RedE ( C ), 5 μ m response regulators RedD ( D ) and RedF ( E ), and their respective point mutants. 32 P-ATP , [γ- 32 P]ATP; 32 P-Ac , [ 32 P]acetyl phosphate; AR , autoradiograph; CS , Coomassie-stained gel.
Figure Legend Snippet: A , domain architecture of RedC–F two-component signaling homologs. Shown is a schematic diagram of conserved domains detected by SMART ( 40 , 41 ) in RedC histidine kinase, RedE histidine kinase-like protein, and RedD and RedF response regulator proteins. Gray rectangles , transmembrane segments predicted by TMpred Server (available on the World Wide Web); white squares , HisKA phosphoaccepting/dimerization domain; white triangles , HATPase_c ATP binding and hydrolysis catalytic domain; white circles , REC receiver domain. Amino acid positions of the invariant histidine ( H ) and aspartic acid ( D ) residues predicted to be the sites of phosphorylation are identified in the relevant domains. B–E , autophosphorylation analysis of Red TCS homologs. Shown are 10 μ m purified histidine kinase RedC-T ( B ) or histidine kinase-like RedE ( C ), 5 μ m response regulators RedD ( D ) and RedF ( E ), and their respective point mutants. 32 P-ATP , [γ- 32 P]ATP; 32 P-Ac , [ 32 P]acetyl phosphate; AR , autoradiograph; CS , Coomassie-stained gel.

Techniques Used: Binding Assay, Purification, Autoradiography, Staining

75) Product Images from "Protein kinase C? regulates phospholipase D activity in rat-1 fibroblasts expressing the ?1A adrenergic receptor"

Article Title: Protein kinase C? regulates phospholipase D activity in rat-1 fibroblasts expressing the ?1A adrenergic receptor

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-5-4

PHE decreases PKCζ activity in rat-1 fibroblasts. A, Cells were treated with 2 μM PHE for 0, 2, 5, 10 and 15 min, lysed and immunoprecipitated with PKCζ antibody for a kinase assay using [γ- 32 P]ATP and a selective peptide substrate as described in Methods. Relative PKCζ activity was expressed as the fold (increase or decrease) of basal. Values are the mean ± S.E. of four independent experiments. * Value significantly different from the basal, p
Figure Legend Snippet: PHE decreases PKCζ activity in rat-1 fibroblasts. A, Cells were treated with 2 μM PHE for 0, 2, 5, 10 and 15 min, lysed and immunoprecipitated with PKCζ antibody for a kinase assay using [γ- 32 P]ATP and a selective peptide substrate as described in Methods. Relative PKCζ activity was expressed as the fold (increase or decrease) of basal. Values are the mean ± S.E. of four independent experiments. * Value significantly different from the basal, p

Techniques Used: Activity Assay, Immunoprecipitation, Kinase Assay

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Autoradiography:

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Construct:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: Protein Expression in Escherichia coli —Constructs for the expression in E. coli of MPK2 as a maltose-binding protein (MBP) fusion and of Pto, Ptoas , Pti1, and the kinase-deficient mutant Pti1(K96N) as GST fusions were described previously ( , , , ). .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences).

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: Various PAK4 constructs were expressed in COS-7 cells and lysed in kinase lysis buffer (50 m m Tris-HCl, pH 7.5, 5 m m MgCl2 , 1% Nonidet P-40, 10% glycerol 150 m m NaCl) with addition of fresh protease inhibitors (0.5 μg/ml leupeptin, 1 m m EDTA, 1 μg/ml pepstatin A, 0.2 m m phenylmethylsulfonyl fluoride) and a serine/threonine protein phosphatase inhibitor mixture (Sigma), followed by immunoprecipitation. .. PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C.

Incubation:

Article Title: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus
Article Snippet: Primer extension The primer f2-rev, which is complementary to +88 to +107 of fibA , was labelled at the 5′-end using [γ-32 P]-ATP (9.25 MBq, Amersham) using T4 polynucleotide kinase (New England Biolabs). .. 0.7 pmol of 32 P-labelled f2-rev and 5 µg of total RNA isolated as described from exponentially growing DK1622 were mixed in 10 µl of hybridization buffer (50 mM HEPES, pH 7.0, 100 mM KCl) and incubated for 1 min at 70°C and slowly cooled to 45°C.

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: .. Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count). .. For in vivo labeling, 293T cells (5 × 106 ) were electroporated with 20 μg of VP40 plasmid, followed by addition of Hygromycin B (200 μg/mL).

Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells
Article Snippet: .. Beads were resuspended in 40 µl PNK reaction mix [4 µl 10X PNK buffer (New England BioLabs), 1 µl [γ-32 P] ATP (GE), 2 µl T4 PNK enzyme (10 U/µl; New England BioLabs), 32 µl water] and incubated at 37 °C for 10 min. .. Samples were washed three times in 200 µl PNK buffer and eluted from beads in a mixture of 15 µl PNK buffer and 15 µl LDS sample buffer (Invitrogen) with heating at 70 °C.

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C. .. Incubation was stopped in Laemmli buffer, and samples were heated at 95 °C for 4 min. Phosphorylated proteins were separated by 12.5% SDS-PAGE.

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate. .. The reactions were terminated after a 30-min incubation at 30°C with the addition of sodium dodecyl sulfate-containing gel loading buffer followed by boiling.

Activity Assay:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences). ..

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: .. PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C. .. Incubation was stopped in Laemmli buffer, and samples were heated at 95 °C for 4 min. Phosphorylated proteins were separated by 12.5% SDS-PAGE.

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
Article Snippet: .. Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ). .. p27+/− mice were crossed with MMTV - neu mice, expressing the neu proto-oncogene ( ) (Jackson Laboratories, Bar Harbor, Maine).

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: The kinase activity of Ime2p-myc was measured as described by Benjamin et al. ( ). .. A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate.

Expressing:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: Expression in E. coli and purification of MBP fusions with amylose resin were performed according to the instructions of the manufacturer (New England Biolabs). .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences).

Modification:

Article Title: Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA
Article Snippet: Dithiothreitol (DTT), isopropyl-β- d -thiogalactopyranoside, lysozyme, restriction and modification enzymes and protease inhibitors (PMSF, Bestatin, Leupeptin and Pepstatin) were from Merck, Calbiochem, Boehringer and Promega, respectively. .. [α-32 P]dATP and [γ-32 P]ATP were from Amersham Corp (Germany).

Western Blot:

Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells
Article Snippet: A sample of the cleared supernatant was retained for Western analysis, and the beads were washed three times in RIPA buffer, then twice in PNK buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgCl2 , 0.5% NP-40). .. Beads were resuspended in 40 µl PNK reaction mix [4 µl 10X PNK buffer (New England BioLabs), 1 µl [γ-32 P] ATP (GE), 2 µl T4 PNK enzyme (10 U/µl; New England BioLabs), 32 µl water] and incubated at 37 °C for 10 min.

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
Article Snippet: The precipitates were either utilized for in vitro kinase assays or resolved by SDS-PAGE and Western analysis. .. Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ).

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: .. A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate. .. The reactions were terminated after a 30-min incubation at 30°C with the addition of sodium dodecyl sulfate-containing gel loading buffer followed by boiling.

Kinase Assay:

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: .. The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver. .. The reaction was carried out at 30°C for 10 min and terminated by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiling the mixture for 5 min.

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Kinase assay with peptides was accomplished by adding 50 μg appropriate peptide [S3 (ADLTSPEKIQAI), S3M (ADLTAPEKIQAI), T7 (GKKVTAKNGQPI), S8 (GKKVASKNGQPI), T2 (LVHKLTGKKV) (Biomatik USA, LLC, Wilmington, DE, USA)] and 15 μL of appropriate antibody (α-CycE, α-IgG) to the Protein A/G pellet suspended in kinase buffer (120 μL). .. Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: Paragraph title: Kinase Activity Assay and Phosphopeptide Mapping ... PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C.

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: Paragraph title: In vitro kinase assay. ... A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate.

Hybridization:

Article Title: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus
Article Snippet: Primer extension The primer f2-rev, which is complementary to +88 to +107 of fibA , was labelled at the 5′-end using [γ-32 P]-ATP (9.25 MBq, Amersham) using T4 polynucleotide kinase (New England Biolabs). .. 0.7 pmol of 32 P-labelled f2-rev and 5 µg of total RNA isolated as described from exponentially growing DK1622 were mixed in 10 µl of hybridization buffer (50 mM HEPES, pH 7.0, 100 mM KCl) and incubated for 1 min at 70°C and slowly cooled to 45°C.

Immunoprecipitation:

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: Paragraph title: Immunoprecipitation and S6K assay. ... The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver.

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Phosphorylation reactions were performed with immunoprecipitated material and E. coli -purified VP40 (0.5 μg) as a substrate in TTK kinase buffer containing 50 mM HEPES (pH 7.9), 10 mM MgCl2 , 6 mM EGTA, and 2.5 mM dithiothreitol. .. Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: Various PAK4 constructs were expressed in COS-7 cells and lysed in kinase lysis buffer (50 m m Tris-HCl, pH 7.5, 5 m m MgCl2 , 1% Nonidet P-40, 10% glycerol 150 m m NaCl) with addition of fresh protease inhibitors (0.5 μg/ml leupeptin, 1 m m EDTA, 1 μg/ml pepstatin A, 0.2 m m phenylmethylsulfonyl fluoride) and a serine/threonine protein phosphatase inhibitor mixture (Sigma), followed by immunoprecipitation. .. PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C.

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
Article Snippet: Paragraph title: Immunoprecipitation and kinase assays. ... Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ).

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: Equal amounts of protein (2 to 6 mg) from clarified supernatants were used for immunoprecipitation of Ime2p-myc with 20 μl of a 1:1 slurry of 9E10 beads (Covance) by mixing for 1.5 h. Immunoprecipitates were collected and washed three times in lysis buffer and once in kinase buffer (20 mM HEPES [pH 7.4], 100 mM KCl, 10 mM MgCl2 ). .. A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate.

Protease Inhibitor:

Article Title: Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3
Article Snippet: [γ-32 P]ATP, ECL® reagent and materials for protein purification were obtained from Amersham Biosciences (Chalfont St Giles, Bucks., U.K.). .. Unlabelled ATP and ‘complete EDTA-free protease inhibitor cocktail’ were from Roche Molecular Biochemicals (Lewes, E. Sussex, U.K.), Precision prestained protein molecular mass markers from Bio-Rad (Hemel Hempstead, Herts., U.K.) and cell culture media, precast Bis-Tris SDS/10% polyacrylamide gels, running buffer and transfer buffer were from Invitrogen (Paisley, Scotland, U.K.).

Cell Culture:

Article Title: Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3
Article Snippet: [γ-32 P]ATP, ECL® reagent and materials for protein purification were obtained from Amersham Biosciences (Chalfont St Giles, Bucks., U.K.). .. Unlabelled ATP and ‘complete EDTA-free protease inhibitor cocktail’ were from Roche Molecular Biochemicals (Lewes, E. Sussex, U.K.), Precision prestained protein molecular mass markers from Bio-Rad (Hemel Hempstead, Herts., U.K.) and cell culture media, precast Bis-Tris SDS/10% polyacrylamide gels, running buffer and transfer buffer were from Invitrogen (Paisley, Scotland, U.K.).

other:

Article Title: Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-? and -?
Article Snippet: [γ-32 P]ATP and 86 Rb were from Amersham Biosciences (Piscataway, NJ).

DNA Sequencing:

Article Title: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus
Article Snippet: Primer extension The primer f2-rev, which is complementary to +88 to +107 of fibA , was labelled at the 5′-end using [γ-32 P]-ATP (9.25 MBq, Amersham) using T4 polynucleotide kinase (New England Biolabs). .. Reaction products were separated on a 6% DNA sequencing gel ( ) and visualized by autoradiography.

Polymerase Chain Reaction:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: A series of successive 10 nucleotide deletions were made to the clone 4–15 RNA template using appropriately designed PCR primers. .. Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs).

Sonication:

Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells
Article Snippet: Samples were sonicated and the resulting lysate was treated with RQ1 DNase (Promega) for 5 min at 37 °C, and then diluted RNase A (USB) was added for an additional 10 min. .. Beads were resuspended in 40 µl PNK reaction mix [4 µl 10X PNK buffer (New England BioLabs), 1 µl [γ-32 P] ATP (GE), 2 µl T4 PNK enzyme (10 U/µl; New England BioLabs), 32 µl water] and incubated at 37 °C for 10 min.

Affinity Purification:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: GST fusion proteins were then affinity-purified by using glutathione-agarose beads (Sigma). .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences).

Recombinant:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences). ..

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: Whole-cell extracts were centrifuged at 10,000 × g for 15 min at 4°C, and recombinant EE-S6Ks were immunoprecipitated with the anti-EE monoclonal antibody immobilized on protein G-Sepharose beads (Amersham Pharmacia Biotech). .. The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver.

Paper Chromatography:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: The radiolabeled RNAs were analyzed using paper chromatography and cellulose-packed column assays as described above. .. Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs).

Nucleic Acid Electrophoresis:

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver. .. The reaction was carried out at 30°C for 10 min and terminated by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiling the mixture for 5 min.

In Vivo:

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Paragraph title: Immunoprecipication, in vivo Labeling, and Kinase Assays ... Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

Mutagenesis:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: Protein Expression in Escherichia coli —Constructs for the expression in E. coli of MPK2 as a maltose-binding protein (MBP) fusion and of Pto, Ptoas , Pti1, and the kinase-deficient mutant Pti1(K96N) as GST fusions were described previously ( , , , ). .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences).

Isolation:

Article Title: The orphan histidine protein kinase SgmT is a c-di-GMP receptor and regulates composition of the extracellular matrix together with the orphan DNA binding response regulator DigR in Myxococcus xanthus
Article Snippet: Primer extension The primer f2-rev, which is complementary to +88 to +107 of fibA , was labelled at the 5′-end using [γ-32 P]-ATP (9.25 MBq, Amersham) using T4 polynucleotide kinase (New England Biolabs). .. 0.7 pmol of 32 P-labelled f2-rev and 5 µg of total RNA isolated as described from exponentially growing DK1622 were mixed in 10 µl of hybridization buffer (50 mM HEPES, pH 7.0, 100 mM KCl) and incubated for 1 min at 70°C and slowly cooled to 45°C.

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: .. The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver. .. The reaction was carried out at 30°C for 10 min and terminated by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiling the mixture for 5 min.

Labeling:

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Paragraph title: Immunoprecipication, in vivo Labeling, and Kinase Assays ... Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs). .. Alternatively, RNAs were 3′ end labeled using T4 RNA ligase and [32 P]pCp (GE Healthcare) using the protocol supplied by the enzyme manufacturer (Ambion).

Purification:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: Expression in E. coli and purification of MBP fusions with amylose resin were performed according to the instructions of the manufacturer (New England Biolabs). .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences).

Article Title: Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA
Article Snippet: The Hbsu and β proteins were purified as described previously ( ). .. [α-32 P]dATP and [γ-32 P]ATP were from Amersham Corp (Germany).

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Phosphorylation reactions were performed with immunoprecipitated material and E. coli -purified VP40 (0.5 μg) as a substrate in TTK kinase buffer containing 50 mM HEPES (pH 7.9), 10 mM MgCl2 , 6 mM EGTA, and 2.5 mM dithiothreitol. .. Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs). .. The radiolabeled RNAs were purified by denaturing PAGE and recovered as described above.

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: .. A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate. .. The reactions were terminated after a 30-min incubation at 30°C with the addition of sodium dodecyl sulfate-containing gel loading buffer followed by boiling.

Protein Purification:

Article Title: Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3
Article Snippet: .. [γ-32 P]ATP, ECL® reagent and materials for protein purification were obtained from Amersham Biosciences (Chalfont St Giles, Bucks., U.K.). .. Unlabelled ATP and ‘complete EDTA-free protease inhibitor cocktail’ were from Roche Molecular Biochemicals (Lewes, E. Sussex, U.K.), Precision prestained protein molecular mass markers from Bio-Rad (Hemel Hempstead, Herts., U.K.) and cell culture media, precast Bis-Tris SDS/10% polyacrylamide gels, running buffer and transfer buffer were from Invitrogen (Paisley, Scotland, U.K.).

Polyacrylamide Gel Electrophoresis:

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver. .. The reaction was carried out at 30°C for 10 min and terminated by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiling the mixture for 5 min.

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs). .. The radiolabeled RNAs were purified by denaturing PAGE and recovered as described above.

SDS Page:

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C. .. Incubation was stopped in Laemmli buffer, and samples were heated at 95 °C for 4 min. Phosphorylated proteins were separated by 12.5% SDS-PAGE.

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
Article Snippet: The precipitates were either utilized for in vitro kinase assays or resolved by SDS-PAGE and Western analysis. .. Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ).

Plasmid Preparation:

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count). .. For in vivo labeling, 293T cells (5 × 106 ) were electroporated with 20 μg of VP40 plasmid, followed by addition of Hygromycin B (200 μg/mL).

Software:

Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction
Article Snippet: Gels were subjected to autoradiography and quantification using PhosphorImager software (Amersham Biosciences). .. Next, [γ-32 P] ATP was added to the samples, incubated at 37°C for 1 h, and samples (10 μL) were dotted onto Whatman glass microfiber filters, dried for 30 min, and subsequently submerged in TE buffer with gentle agitation for 48 h. Samples were quantified using a scintillation counter (QuantaSmartTM DPM assay count).

In Vitro:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: .. Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences). ..

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Internally 32 P-labeled RNAs were produced by in vitro transcription of the resulting double-stranded DNAs in the presence of [α-32 P]ATP. .. Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs).

Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells
Article Snippet: The precipitates were either utilized for in vitro kinase assays or resolved by SDS-PAGE and Western analysis. .. Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ).

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: Paragraph title: In vitro kinase assay. ... A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate.

Produced:

Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION
Article Snippet: Internally 32 P-labeled RNAs were produced by in vitro transcription of the resulting double-stranded DNAs in the presence of [α-32 P]ATP. .. Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs).

Concentration Assay:

Article Title: Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands *Bypassing Kinase Activity of the Tomato Pto Resistance Protein with Small Molecule Ligands * S⃞
Article Snippet: Kinase Activity Assays —Kinase assays to test autophosphorylation of Pto forms, Pti1, and MPK2 were performed in vitro with 2 μg of the recombinant kinase in 20 μl of kinase reaction buffer (50 m m Tris-HCl, pH 7.0, 1 m m dithiothreitol, 10 m m MnCl2 , and 20 m m ATP) containing 1 μCi of [γ-32 P]ATP (3,000 Ci/mmol; Amersham Biosciences). .. PP1 analogs were synthesized as described ( , ) and their effect on kinase activity was tested at the final concentration of 1 μ m .

Article Title: Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA
Article Snippet: [α-32 P]dATP and [γ-32 P]ATP were from Amersham Corp (Germany). .. The concentration of DNA was determined using a molar extinction coefficient of 6500 M–1 cm–1 at 260 nm.

Lysis:

Article Title: Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ?II and Regulates Its Subcellular Localization
Article Snippet: Immune complexes were washed three times with lysis buffer followed by a single wash with kinase assay buffer (50 mM HEPES [pH 7.5], 10 mM MgCl2 , 1 mM dithiothreitol, 10 mM β-glycerophosphate). .. The kinase reaction was initiated by resuspending the beads in 25 μl of kinase assay buffer supplemented with 1 μM protein kinase A inhibitor (Calbiochem), 50 μM ATP, 5 μCi of [γ-32 P]ATP (Amersham Pharmacia Biotech), and 20 μg of 40S ribosomes isolated from rat liver.

Article Title: p21-activated Kinase 4 Phosphorylation of Integrin ?5 Ser-759 and Ser-762 Regulates Cell Migration *
Article Snippet: Various PAK4 constructs were expressed in COS-7 cells and lysed in kinase lysis buffer (50 m m Tris-HCl, pH 7.5, 5 m m MgCl2 , 1% Nonidet P-40, 10% glycerol 150 m m NaCl) with addition of fresh protease inhibitors (0.5 μg/ml leupeptin, 1 m m EDTA, 1 μg/ml pepstatin A, 0.2 m m phenylmethylsulfonyl fluoride) and a serine/threonine protein phosphatase inhibitor mixture (Sigma), followed by immunoprecipitation. .. PAK4 kinase activity was determined in a kinase buffer (50 m m Hepes, pH 7.5, 10 m m MgCl2 , 2 m m MnCl2 , 0.2 m m dithiothreitol) in the presence of 30 μ m cold ATP and 10 μCi of [γ-32 P]ATP (3000 Ci/n m , Amersham Biosciences) and in the presence of 5 μg of substrate (MBP, GST, GST-β1 tail, or GST-β5 tail) for 30 min at 30 °C.

Article Title: The Cdk-Activating Kinase Cak1p Promotes Meiotic S Phase through Ime2p †
Article Snippet: Equal amounts of protein (2 to 6 mg) from clarified supernatants were used for immunoprecipitation of Ime2p-myc with 20 μl of a 1:1 slurry of 9E10 beads (Covance) by mixing for 1.5 h. Immunoprecipitates were collected and washed three times in lysis buffer and once in kinase buffer (20 mM HEPES [pH 7.4], 100 mM KCl, 10 mM MgCl2 ). .. A third of the immunoprecipitate was removed for Western blotting, and the remainder was resuspended in 25 μl of kinase buffer plus 5 μCi of [γ-32 P]ATP (Amersham), 10 μM ATP, and 10 μg of purified MBP-Ndt80p substrate.

Cross-linking Immunoprecipitation:

Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells
Article Snippet: Paragraph title: Formaldehyde cross-linking immunoprecipitation ... Beads were resuspended in 40 µl PNK reaction mix [4 µl 10X PNK buffer (New England BioLabs), 1 µl [γ-32 P] ATP (GE), 2 µl T4 PNK enzyme (10 U/µl; New England BioLabs), 32 µl water] and incubated at 37 °C for 10 min.

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    GE Healthcare γ 32 p atp
    RNA is purified from cell lysates by anti-p53 antibodies regardless of p53 status. (A) Western analysis of p53. Preparations of whole cell lysate (WCL; 10% of total) [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4, after IP with anti-p53 Do-7 (lanes 5–8, respectively). (B) Co-immunoprecipitated RNA after [γ 32 <t>P]-ATP</t> labeling: HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4. Formaldehyde cross-linked whole cell lysate: [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4], subjected to IP with anti-p53 Do-7 followed by RNA radiolabeling. (C) Western blot with anti-p53 antibody Do-1. (D) Membrane from panel (C) exposed to film.
    γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNA is purified from cell lysates by anti-p53 antibodies regardless of p53 status. (A) Western analysis of p53. Preparations of whole cell lysate (WCL; 10% of total) [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4, after IP with anti-p53 Do-7 (lanes 5–8, respectively). (B) Co-immunoprecipitated RNA after [γ 32 P]-ATP labeling: HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4. Formaldehyde cross-linked whole cell lysate: [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4], subjected to IP with anti-p53 Do-7 followed by RNA radiolabeling. (C) Western blot with anti-p53 antibody Do-1. (D) Membrane from panel (C) exposed to film.

    Journal: Biochemical and biophysical research communications

    Article Title: Analysis of p53-RNA Interactions in Cultured Human Cells

    doi: 10.1016/j.bbrc.2007.08.181

    Figure Lengend Snippet: RNA is purified from cell lysates by anti-p53 antibodies regardless of p53 status. (A) Western analysis of p53. Preparations of whole cell lysate (WCL; 10% of total) [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4, after IP with anti-p53 Do-7 (lanes 5–8, respectively). (B) Co-immunoprecipitated RNA after [γ 32 P]-ATP labeling: HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4. Formaldehyde cross-linked whole cell lysate: [HCT116 ( tp53 −/−) lane 1; HCT116 ( TP53 +/+) lane 2; PC-3 ( tp53 −/−) lane 3; MCF-7 ( TP53 +/+) lane 4], subjected to IP with anti-p53 Do-7 followed by RNA radiolabeling. (C) Western blot with anti-p53 antibody Do-1. (D) Membrane from panel (C) exposed to film.

    Article Snippet: Beads were resuspended in 40 µl PNK reaction mix [4 µl 10X PNK buffer (New England BioLabs), 1 µl [γ-32 P] ATP (GE), 2 µl T4 PNK enzyme (10 U/µl; New England BioLabs), 32 µl water] and incubated at 37 °C for 10 min.

    Techniques: Purification, Western Blot, Immunoprecipitation, Labeling, Radioactivity

    Minimization of clone 4–15 cellulose-binding RNA. (A) Locations of a series of successive 10 nucleotide truncations (TR1 through TR6) used to assess the minimal length of the clone 4–15 aptamer that retains function. Shaded nucleotides reflect the minimal functional RNA derived from the data depicted in Fig. 3C. (B) Paper chromatogram assessing the cellulose-binding functions of various 32 P-labeled RNA constructs. Random-sequence G0 RNAs are used as a control. (C) Single-nucleotide-resolution mapping of boundaries of the cellulose-binding RNA aptamer 4–15. RNAs were labeled on 5′ or 3′ termini using [γ- 32 P]ATP or [ 32 P]pCp, respectively. Radiolabeled RNAs were subjected to partial digestion with alkali and then were applied to cellulose-packed columns. RNA fragments retained by the cellulose matrix were recovered using elution buffer (lanes 1 and 3). RNAs that were unable to bind to cellulose were also collected (lanes 2 and 4).

    Journal: Nucleosides, nucleotides & nucleic acids

    Article Title: IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    doi: 10.1080/15257770802257903

    Figure Lengend Snippet: Minimization of clone 4–15 cellulose-binding RNA. (A) Locations of a series of successive 10 nucleotide truncations (TR1 through TR6) used to assess the minimal length of the clone 4–15 aptamer that retains function. Shaded nucleotides reflect the minimal functional RNA derived from the data depicted in Fig. 3C. (B) Paper chromatogram assessing the cellulose-binding functions of various 32 P-labeled RNA constructs. Random-sequence G0 RNAs are used as a control. (C) Single-nucleotide-resolution mapping of boundaries of the cellulose-binding RNA aptamer 4–15. RNAs were labeled on 5′ or 3′ termini using [γ- 32 P]ATP or [ 32 P]pCp, respectively. Radiolabeled RNAs were subjected to partial digestion with alkali and then were applied to cellulose-packed columns. RNA fragments retained by the cellulose matrix were recovered using elution buffer (lanes 1 and 3). RNAs that were unable to bind to cellulose were also collected (lanes 2 and 4).

    Article Snippet: Full-length clone 4–15 RNAs were dephosphorylated using calf intestinal phosphatase (Roche) and subsequently 5′ 32 P-labeled using T4 polynucleotide kinase and [γ-32 P]ATP (GE Healthcare) following the protocol supplied by the enzyme manufacturer (New England Biolabs).

    Techniques: Binding Assay, Functional Assay, Derivative Assay, Labeling, Construct, Sequencing

    Phosphorylation and activation of nSMase1 by JNK in vitro . ( a ) The serine-270 phosphorylation of nSMase1 by JNK in vitro . Mouse JNK1 was used for an in vitro kinase assay using [ γ - 32 P]-ATP and either nSMase1 (wild-type) or mutant protein (S270A) as the substrate. The reactions were incubated at 30 °C for 30 min with or without 2 ng of mouse JNK1 and in the presence (+) or absence (−) of 10 μ M SP600125. Recombinant proteins from each reaction were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Phosphorylated nSMase1 was detected by autoradiography (upper panel). The levels of substrate protein present in each reaction was determined using Coomassie Brilliant Blue R-250 staining (lower panel). ( b ) The effect of phosphorylation on the activation of nSMase1. The nSMase activity in each column is shown. After the in vitro kinase assay, the postreaction mixture was analyzed in an nSMase enzymatic assay using C 6 -NBD-sphingomyelin (black columns). White columns indicate the basal enzyme activity before the kinase assay. The basal enzyme activities of the recombinant nSMase1 (wild-type) and nSMase1 mutant (S270A) were 22.4±0.48 and 22.6±0.35 μ mol/mg/h, respectively. Column 1, nSMase1 wild-type; column 2, nSMase1 treated with JNK; column 3, nSMase1 mutant (S270A) treated with JNK; column 4, nSMase1 treated with JNK in the presence of a JNK inhibitor. The enzyme activity after JNK treatment (black column 2) was 120.1±21.4 μ mol/mg/h. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. * P

    Journal: Cell Death and Differentiation

    Article Title: Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

    doi: 10.1038/cdd.2014.128

    Figure Lengend Snippet: Phosphorylation and activation of nSMase1 by JNK in vitro . ( a ) The serine-270 phosphorylation of nSMase1 by JNK in vitro . Mouse JNK1 was used for an in vitro kinase assay using [ γ - 32 P]-ATP and either nSMase1 (wild-type) or mutant protein (S270A) as the substrate. The reactions were incubated at 30 °C for 30 min with or without 2 ng of mouse JNK1 and in the presence (+) or absence (−) of 10 μ M SP600125. Recombinant proteins from each reaction were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Phosphorylated nSMase1 was detected by autoradiography (upper panel). The levels of substrate protein present in each reaction was determined using Coomassie Brilliant Blue R-250 staining (lower panel). ( b ) The effect of phosphorylation on the activation of nSMase1. The nSMase activity in each column is shown. After the in vitro kinase assay, the postreaction mixture was analyzed in an nSMase enzymatic assay using C 6 -NBD-sphingomyelin (black columns). White columns indicate the basal enzyme activity before the kinase assay. The basal enzyme activities of the recombinant nSMase1 (wild-type) and nSMase1 mutant (S270A) were 22.4±0.48 and 22.6±0.35 μ mol/mg/h, respectively. Column 1, nSMase1 wild-type; column 2, nSMase1 treated with JNK; column 3, nSMase1 mutant (S270A) treated with JNK; column 4, nSMase1 treated with JNK in the presence of a JNK inhibitor. The enzyme activity after JNK treatment (black column 2) was 120.1±21.4 μ mol/mg/h. Each value represents the mean of three independent experiments, and the error bars represent the S.D.s. * P

    Article Snippet: [γ -32 P]-ATP (370 MBq mM/ml), Hybond-P polyvinylidene fluoride (PVDF) membrane, protein G-Sepharose, and secondary antibodies were acquired from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Activation Assay, In Vitro, Kinase Assay, Mutagenesis, Incubation, Recombinant, Autoradiography, Staining, Activity Assay, Enzymatic Assay

    Insulin stabilizes a set of KSRP-interacting mRNAs. (A) Serum-starved HIRc-B cells were treated for 1 h with either PBS (control) or insulin (10 -6 M). Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) In vitro RNA degradation assays using S100 extracts from either control or insulin (10 -6 M)-treated HIRc-B cells. Internally 32 P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with S100 extracts for the indicated times and their decay analyzed as described in Methods.

    Journal: BMC Molecular Biology

    Article Title: Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

    doi: 10.1186/1471-2199-8-28

    Figure Lengend Snippet: Insulin stabilizes a set of KSRP-interacting mRNAs. (A) Serum-starved HIRc-B cells were treated for 1 h with either PBS (control) or insulin (10 -6 M). Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) In vitro RNA degradation assays using S100 extracts from either control or insulin (10 -6 M)-treated HIRc-B cells. Internally 32 P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with S100 extracts for the indicated times and their decay analyzed as described in Methods.

    Article Snippet: [γ-32 P]ATP (3000 Ci/mmol) was from GE Healthcare.

    Techniques: Immunoprecipitation, Incubation, Labeling, SDS Page, Autoradiography, In Vitro

    PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression. (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 7 .

    Journal: BMC Molecular Biology

    Article Title: Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

    doi: 10.1186/1471-2199-8-28

    Figure Lengend Snippet: PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression. (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[ 32 P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 7 .

    Article Snippet: [γ-32 P]ATP (3000 Ci/mmol) was from GE Healthcare.

    Techniques: Expressing, Immunoprecipitation, Incubation, Labeling, SDS Page, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Quantitation Assay