na-pp1 (Merck KGaA)
90
Structured Review
Merck KGaA
na-pp1
Na Pp1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+reaction+buffer/na+pp1/pmc03418839-285-55-56
Average 90 stars, based on 1 article reviews
Na Pp1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pp1+reaction+buffer/na+pp1/pmc03418839-285-55-56
Average 90 stars, based on 1 article reviews
na-pp1 - by Bioz Stars,
2026-11
90/100 stars
Images
1) Product Images from "Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex * "
Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex
Journal: Molecular & Cellular Proteomics : MCP
doi: 10.1074/mcp.M111.013912
Figure Legend Snippet: The H250Y mutation rescues kinase activity of Aurora B gatekeeper mutants. A, multiple sequence alignment of the Aurora kinases in different species. The gatekeeper residue and the second site suppressor mutation found in Ark1 and Ipl1 as well as the newly identified second site suppressor mutation for human and mouse Aurora B are indicated. B, in vitro kinase assay with the indicated recombinant GST-tagged Aurora B proteins and recombinant histone H3 as substrate. Because one master mix of kinase reaction buffer plus histone H3 was used, each reaction was supplemented with the same amount of substrate. Phosphorylation of histone H3 was detected with an antibody specific for phosphoserine 10. Note that even a small amount of Aurora BH250Y results in massive phosphorylation of histone H3. C, mitotic index of paclitaxel-treated U2OS cells transfected with cDNAs encoding the indicated Aurora B mutants. All three mutants are dominant negative over the endogenous kinase and therefore reduce the mitotic delay induced by paclitaxel. D and E, FLAG-tagged human Aurora B mutants (D) and GFP-tagged mouse Aurora B mutants (E) were immunoprecipitated from mitotic U2OS cells. Kinase reactions were performed in the presence of ATP and with or without NA-PP1 (2 μm). Histone H3 was used as substrate. F, the H250Y mutation rescues kinase activity of Aurora B gatekeeper mutants. The double mutants are inhibited by NA-PP1.
Techniques Used: Mutagenesis, Activity Assay, Sequencing, In Vitro, Kinase Assay, Recombinant, Transfection, Dominant Negative Mutation, Immunoprecipitation
Figure Legend Snippet: The analog-sensitive Aurora B mutants are inhibited by PP1 analogs in cells. A, U2OS cells expressing the indicated proteins were released from a thymidine-induced G1/S block into medium containing paclitaxel plus or minus the indicated PP1 analogs. Seventeen hours after release, the mitotic index was determined by propidium iodide/MPM2 monoclonal antibody labeling and FACS analysis. B, similar to A, but now different concentrations of NA-PP1 were added to the cells. For each transfected cell population, the mitotic index without PP1 inhibitor was set to 100%, and the relative reduction in mitotic index in the presence of PP1 inhibitor was determined.
Techniques Used: Expressing, Blocking Assay, Antibody Labeling, Transfection
Figure Legend Snippet: Localization of the analog-sensitive Aurora B mutants. U2OS cells expressing the indicating GFP-tagged proteins were fixed 14 h after release from a thymidine-induced G1/S block into the Eg5 inhibitor S-trityl-l-cysteine (44) with or without NA-PP1 (2 μm). Centromeres were detected with human CREST antiserum and endogenous survivin with anti-survivin polyclonal antibody. DNA was counterstained with 4′,6′-diamino-2-phenylindole (DAPI). Note the displacement over the chromosomal arms when kinase activity is inhibited by NA-PP1.
Techniques Used: Expressing, Blocking Assay, Activity Assay
Figure Legend Snippet: The Aurora B analog-sensitive mutant thiophosphorylates multiple proteins in cell extracts. A, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B, co-purified from SF9 cells, in the presence of ATP and with or without NA-PP1 (2 μm). Histone H3 was used as a substrate. B, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B (wild-type or analog-sensitive mutants) in the presence of ATPγS, N6-furfuryl-ATPγS (Fu), N6-phenylethyl-ATPγS (Phe), or N6-benzyl-ATPγS (Bn). Thiophosphorylated histone H3 was detected with a thiophosphate ester epitope-specific antibody (13). C, in vitro kinase assay with His-INCENP/GST-Aurora BL154G/H250Y in the presence of N6-furfuryl-ATPγS. 50 μg of a mitotic HeLa cell lysate was used as input, and histone H3 was spiked into this lysate to serve as positive control. For the lower part of the blot (containing proteins with molecular masses of 15–50 kDa), a longer exposure (5 min instead of 15 s for the short exposure) is shown.
Techniques Used: Mutagenesis, In Vitro, Kinase Assay, Recombinant, Purification, Positive Control
Related Articles
Immunoprecipitation:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Recombinant:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Mutagenesis:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Activity Assay:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Sequencing:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM In Vitro:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Kinase Assay:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Transfection:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Dominant Negative Mutation:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Expressing:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Blocking Assay:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Antibody Labeling:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Purification:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM Positive Control:Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex Article Snippet: The cells were washed two times in PBS with 0.05% Tween 20 and incubated with mouse anti-MPM2 (Upstate Biotechnology) as primary antibody and goat anti-mouse Cy5 (Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Article Title: Analog-sensitive cell line identifies cellular substrates of CDK9 Article Snippet: 1 × 10 7 cells were treated with 10 μM |
