recombinant chk2 kinase  (R&D Systems)

Journal: Nucleic Acids Research

Article Title: Phosphorylation and ubiquitination-dependent degradation of CABIN1 releases p53 for transactivation upon genotoxic stress

doi: 10.1093/nar/gks1319

Figure Lengend Snippet: CABIN1 is phosphorylated by DNA damage kinase. ( A ) HEK293 cells were transfected with the indicated combination of Myc-tagged CABIN1 and FLAG-tagged ATM. To test the physical interaction, immunoprecipitation assays were performed and analyzed by immunoblotting. ( B ) A stable cell line expressing FLAG-tagged CABIN1 was used for recombinant FLAG-CABIN1 purification. Purified FLAG-tagged CABIN1 protein was resolved by SDS–PAGE and stained with Coomassie blue. ( C ) HEK293 cells were transfected with FLAG-ATM wild-type or kinase dead mutants. Using IP-ed FLAG-ATM from transiently transfected cells and purified recombinant proteins, in vitro kinase assay was performed and assessed by autoradiography. The middle panel shows Coomassie staining of CABIN1 and the bottom panel shows the immunoblotting results of ectopically expressed FLAG-ATM wild-type and kinase dead mutants. ( D ) Cells were transfected with the combination of Myc-tagged CABIN1 and FLAG-tagged CHK2. Lysates from the transfectants were subjected to immunoprecipitation with FLAG antibody and were analyzed by immunoblotting with the indicated antibodies. ( E ) CHK2 kinase assay was performed using purified recombinant proteins. Phosphorylated CABIN1 was visualized by autoradiography (upper panel) and phosphorylated CHK2 was also detected as a positive control (bottom panel).

Article Snippet: For CHK2 kinase assay, the purified recombinant full-length human CHK2 kinase (#7434, Cell Signaling Technology) was used following the manufacturer’s protocol.

Techniques: Transfection, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Recombinant, Purification, SDS Page, Staining, In Vitro, Kinase Assay, Autoradiography, Positive Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation and ubiquitination-dependent degradation of CABIN1 releases p53 for transactivation upon genotoxic stress

doi: 10.1093/nar/gks1319

Figure Lengend Snippet: Inhibition of DNA damage kinase activities blocks CABIN1 degradation upon DNA damage. ( A ) HCT116 cells were UV-irradiated with or without caffeine and harvested 4 h later. The CABIN1 protein levels were checked by immunoblotting. ( B and D ) Using the lentiviral shRNA knockdown system, ATM or CHK2 was reduced and the indicated protein levels were checked under UV irradiation condition. ( C ) Cells were UV-irradiated with or without CHK2 inhibitor II and then, the CABIN1 protein levels were determined by immunoblotting.

Article Snippet: For CHK2 kinase assay, the purified recombinant full-length human CHK2 kinase (#7434, Cell Signaling Technology) was used following the manufacturer’s protocol.

Techniques: Inhibition, Irradiation, Western Blot, shRNA, Knockdown

Journal: Nucleic Acids Research

Article Title: Phosphorylation and ubiquitination-dependent degradation of CABIN1 releases p53 for transactivation upon genotoxic stress

doi: 10.1093/nar/gks1319

Figure Lengend Snippet: Schematic model for CABIN1 degradation mechanism upon DNA damage. CABIN1 acts as a repressive regulator of p53 by co-occupying a subset of p53 target promoters in the unstressed condition. Under the genotoxic stress condition, CABIN1 is phosphorylated by ATM and CHK2 and undergoes ubiquitin-dependent proteasomal degradation mediated by the CRL4DDB2 ubiquitin ligase complex.

Article Snippet: For CHK2 kinase assay, the purified recombinant full-length human CHK2 kinase (#7434, Cell Signaling Technology) was used following the manufacturer’s protocol.

Techniques: Ubiquitin Proteomics