human full length bubr1  (Thermo Fisher)

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: C-MAD2 prolongs the lag in APC/C activation in mitotic cell extracts. A, mitotic extracts prepared from checkpoint-active HeLa cells and incubated in the absence (control) or presence of untagged recombinant MAD2L13A. MAD2L13A was added at 2× and 20× above the concentration of endogenous MAD2 in the extract (∼23 nm). APC/C activity was monitored by degradation of endogenous cyclin B1 and securin. Samples were taken at the indicated minutes after incubation and analyzed by immunoblotting. ATM and an APC/C subunit CDC27 were loading controls. B, quantitation of cyclin B1 degradation shown in A. Cyclin B1 levels were quantified and normalized against ATM levels in scanned Western blots, and the mean ± S.D. (error bars) from two experiments were plotted versus time. C, mitotic extracts incubated in the absence (control) or presence of 20× recombinant MAD2L13A and samples at different times and immunoprecipitated (IP) with anti-CDC27 antibody and probed for MCC and APC/C subunits. D, quantitation of experiment shown in C. BUBR1 and MAD2 levels were normalized against CDC16 (another APC/C subunit) in CDC27 immunoprecipitates and were plotted versus time. The level at the 0 time point in the control experiment was set at 100%. E, kinetics of cyclin B1 degradation in mitotic extracts prepared from untransfected cells (control) or cells transfected with GST-MAD2L13A. GST-MAD2 and endogenous MAD2 were detected simultaneously for comparison of levels. F, untagged recombinant MAD2WT and MAD2ΔC10 added to the mitotic extracts and the rates of cyclin B1 degradation compared.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Activation Assay, Incubation, Recombinant, Concentration Assay, Activity Assay, Western Blot, Quantitation Assay, Immunoprecipitation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: The R133E/Q134A mutant reduces C-MAD2 binding to BUBR1 but not CDC20. A, purified recombinant proteins were incubated at intracellular concentrations of endogenous proteins (Fig. 3 legend) for 1 h. GST pulldowns and inputs were probed with the indicated antibodies. Both GST-BUBR1(1–371) and GST-CDC20(111–138) were detected with anti-GST antibody. B, comparison of the full-length BUBR1 binding to MAD2L13A and MAD2LARQ is shown. C, human BUBR1 structure and two models of MCC architecture are illustrated schematically. The two CDC20 binding domains of BUBR1 are shown as CDC20 BD1 and CDC20 BD2. One or both KEN boxes in the N-terminal region of BUBR1 (1–371 residues) are required for CDC20 binding. The GLEBS motif is required for BUBR1-BUB3 interaction. The αC helix of C-MAD2 is represented by a zigzag line.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Mutagenesis, Binding Assay, Purification, Recombinant, Incubation

Journal: The Journal of Biological Chemistry

Article Title: BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

doi: 10.1074/jbc.M111.238543

Figure Lengend Snippet: BUBR1 directly binds to C-MAD2. A, purified recombinant proteins were incubated at the indicated -fold over previously reported intracellular concentrations ([BUBR1] = 127 nm; [CDC20] = 285 nm; [MAD2] = 230 nm]) (24). GST pulldowns were washed and probed alongside inputs with the indicated antibodies. B, comparison of full-length BUBR1 and BUBR1(1–371) in binding to MAD2L13A is shown. C, HeLa cells co-transfected with GST-MAD2W75A and either GFP or GFP-BUBR1(1–371) were arrested in mitosis with nocodazole. The lysates and GST pulldowns were probed for CDC20, MAD1, GST-MAD2, GFP, and GFP-BUBR1(1–371). D, yeast two-hybrid assay was performed. The yeast strain SKY48 harboring different combinations of baits and preys were tested for growth on galactose-containing leucine-dropout plates. The arrowheads indicate the combinations that produced colonies. E, recombinant MAD2WT was preincubated with GST-CDC20(111–138) immobilized on GSH-agarose beads (+) or beads alone (−) for 24 h. After incubation, GSH-agarose beads were removed by centrifugation, and the resulting supernatants were transferred to new binding reactions containing GST-BUBR1(1–371). GST pulldowns and inputs for the new reactions were probed with the indicated antibodies.

Article Snippet: Human full-length BUBR1, MAD2, and CDC20 cDNAs were amplified from a prostate cDNA library (Invitrogen) or freshly prepared reverse transcribed cDNAs provided by Dr. Douglas Leaman (University of Toledo).

Techniques: Purification, Recombinant, Incubation, Binding Assay, Transfection, Y2H Assay, Produced, Centrifugation