Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores

Science

Volumn 274, Issue 5285, 1996, Pages 242-246

  Chen, Rey Huei ^a   Waters, Jennifer C ^a   Salmon, E D ^a   Murray, Andrew W ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

BENZIMIDAZOLE; CELL EXTRACT; COMPLEMENTARY DNA; NOCODAZOLE; PROTEIN;

ANAPHASE; ANIMAL CELL; ARTICLE; CELL CYCLE; CELL DIVISION; CELL LINE; CENTROMERE; CONTROLLED STUDY; FROG; FUNGUS MUTANT; HELA CELL; HUMAN; HUMAN CELL; METAPHASE; MICROTUBULE ASSEMBLY; MITOSIS INHIBITION; MITOSIS SPINDLE; NONHUMAN; OOCYTE; PRIORITY JOURNAL; SACCHAROMYCES CEREVISIAE; YEAST;

AMINO ACID SEQUENCE; ANIMALS; ATP-BINDING CASSETTE TRANSPORTERS; CALCIUM; CELL CYCLE; CELLS, CULTURED; HELA CELLS; HUMANS; INTERPHASE; KINETOCHORES; LAMINS; MICROTUBULES; MITOSIS; MITOTIC SPINDLE APPARATUS; MOLECULAR SEQUENCE DATA; NUCLEAR ENVELOPE; NUCLEAR PROTEINS; OVUM; P-GLYCOPROTEIN; P-GLYCOPROTEINS; PROTAMINE KINASE; XENOPUS;

ANIMALIA; ANURA; FUNGI; METAZOA; SACCHAROMYCES CEREVISIAE; SACCHAROMYCETALES;

EID: 0029845891     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5285.242     Document Type: Article

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23. Affinity purification of anti-XMAD2 was done as described (27). The control antibody, immunoglobulin G (IgG), was purified from preimmune serum over a protein A column made from Affi-Prep Protein A (Bio-Rad 156-0006) as described [E. Harlow and D. Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988)]. Purified antibodies were dialyzed and concentrated in antibody dilution buffer [10 mM sodium phosphate (pH 7.4), 100 mM potassium chloride, 1 mM magnesium chloride, 50% glycerol (v/v)].
24. XTC cells were maintained at 28°C in 70% L-15 medium (Gibco) supplemented with fetal bovine serum (FBS) (10%), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Pen/Strep). HeLa cells were grown in Dulbecco's modified Eagle's medium [with glucose (4.5 g/liter), Gibco] containing 10% FBS and Pen/Strep. Cell lysates were prepared by resuspending the cell pellets or diluting the egg extracts with 10 volumes of lysis buffer [10 mM potassium phosphate (pH 7.5); 1 mM EDTA; 1 mM EGTA; 10 mM magnesium chloride; 50 mM β-glycerophosphate; 1 mM sodium orthovanadate; 1% Triton X-100; 1 mM phenylmethylsulfonyl fluoride; and leupeptin, pepstatin, and chymostatin (each at 10 μg/ ml)] on ice for 10 min followed by centrifugation at 10,000g for 5 min at 4°C. Proteins (20 μg) from the supernatants were loaded in each lane and an immunoblot was prepared with ECL (Amersham).
25. 2-terminus and expressed it in Escherichia coll with Qiaexpress vector pQE10 (Qiagen). The protein was purified according to the manufacturer's instructions. The purified proteins were dialyzed against a buffer containing 50 mM Hepes (pH 7.4), 50 mM potassium chloride, and 50% glycerol.
26. Freshly prepared CSF-arrested extracts (20 μl) were first incubated for 1 hour on ice with 1 μl (0.7 μg) of various antibodies. To block anti-XMAD2, we incubated the antibodies with 6H-XMAD2 (antibody:protein = 3:2 by weight or 1:2 in molar ratio) on ice for 1 hour. The extracts were then incubated with a low or high concentration (3000 or 10,000 nuclei per microliter, respectively) of demembranated sperm nuclei prepared as described (22) for 10 min at room temperature and for another 10 min at room temperature with nocodazole (10 μg/ml). The nocodazole stock was made in dimethyl sulfoxide at 10 mg/ml and was diluted 1:50 (200 μg/ml) in CSF-arrested extract before it was added to the reaction mixture, CSF-arrested extracts were incubated with a high concentration of sperm nuclei and nocodazole for 20 min and then with control antibodies or anti-XMAD2 at room temperature for 30 min. Calcium chloride (0.4 to 0.6 mM) was added to induce exit from mitosis and 1-μl samples were taken every 15 min and left on dry ice until all of the samples were ready for histone H1 kinase activity measurement as described (22).
27. XTC cells were fixed in 3% paraformaldehyde, and immunofluorescent staining was done as described (21), except that all buffers were made in 70% phosphate-buffered saline (PBS). For detergent extraction, cells were first extracted with 70% PBS with 0.5% Triton X-100 (PBST) for 5 min followed by fixation with paraformaldehyde. The images were viewed with a Nikon Microphot-FXA microscope and photographed with Kodak Tri-X pan 400 film.
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29. Cells were first simultaneously extracted and fixed with 0.5% Triton X-100, 2% formaldehyde in PHEM [60 mM Pipes, 25 mM Hepes (pH 7), 10 mM EGTA, 2 mM magnesium chloride] for 5 min and then fixed further in 4% formaldehyde in PHEM for 15 min. Immunofluorescent staining was then performed as described (79), except that the buffer PBS plus 0.05% Tween 20 was used. Z-series optical sections (0.5 μm thick) were captured with a multimode digital fluorescence microscope system. A Nikon Microphot-FXA microscope equipped with a ×60, numerical aperture 1.4 objective was used.
30. We thank A. Desai and T. Mitchison for the CREST serum and for their expertise and encouragement; J. Minshull for the Xenopus ovary cDNA library; R. Benezra, Y, Li, and T. Gustafson for sharing unpublished results; and T. Mitchison, D. Morgan, A. Desai, and members of A.W.M.'s laboratory for their helpful comments on the manuscript. Supported by grants from NIH, the Packard Foundation, the Markey Foundation, and the March of Dimes to A.W.M. R.-H.C. is a Helen Hay Whitney postdoctoral fellow.