The maintenance . Metaphase II arrested extracts were 10,000 nuclei / l erg complements And incubated for 30 min with DMSO or nocodazole at 21 Calcium was then added to sen mitotic exit foreign. Samples were taken before and after the addition of calcium and to determine for cdc2 H1 kinase activity T or blotted with Aurora-A-Antique Alvocidib CDK inhibitor Body. For experiments with the establishment of control points The ‘M 20 ZMwas added to metaphase II extracts on ice and incubated for 30 min. The extracts were then to 21 exp Rmt, complements a With 10,000 nuclei / l and nocodazole and incubated for another 30th Calcium was then added and the samples were removed and analyzed above. Experiences for the maintenance of the control points On, were metaphase II extracts with 10,000 nuclei / l erg Complements and incubated with nocodazole for 30 min at 21 to Checkpoint to induce activation.
ZM was then added to a final concentration of 20 M and extracts were incubated for another 30 min. Calcium was Gamma Secretase pathway added and the samples were taken and analyzed above. excess mitotic arrest in MAD2 causes ZM-treated extracts. Metaphase II arrested extracts were 10,000 nuclei / l erg complements And incubated for 30 min at 21 in the absence or presence of excess Mad2 protein, or in the presence of nocodazole. At the indicated time points samples were taken before and after the addition of calcium will be absorbed and used to determine that cdc2 H1 kinase activity of t. Metaphase II extracts were also treated with 20 M ZM incubated on ice for 30 min and then heated to 21 These extracts were then seeded and erg nocodozole complements And incubated in the absence or presence of excess Mad2 protein for 30 min.
Calcium was added and the samples were removed and used cdc2 H1 kinase activity of t determined. BB Gadea and JV Ruderman 1314 Molecular Biology of the Cell, which is responsible for establishing and maintaining the spindle checkpoint, but only the kinase activity of t is required to establish the checkpoint On. SO K Nnte the addition of Aurora B antibody Rpern st Ren or adversely Mighty protein-protein interactions that are required to have to wait checkpoint, whereas inhibition of kinase activity t of ZM erm Aligned k Nnten these interactions occur. ZM st rt Arrest the spindle checkpoint integrity T at a point upstream of MAD2 kinetochore will only be considered in order to generate the activated form of Mad2, a component of the spindle checkpoint assembly.
Activated Mad2 inhibits APC activator / C Cdc20, an event as the last step in the direction of the spindle checkpoint is. Adding big s amounts of protein in Xenopus extracts Mad2 inhibits APC / C independent Ngig of the kinetochore attachment and signaling. As expected, the addition of an excess Mad2 protein or nocodazole prevented arrest in metaphase II extracts contain a high number of nuclei mitotic exit. As mentioned initially incubated with extract First with ZM and then with nocodazole vers umt, Make the arrest checkpoint Am. However, when extracts were initially Highest incubated with ZM and then with Mad2, the extract remained arrested in mitosis. Thus ZM seems a step that occurs prior to inhibit Mad2. The main points to emerge from this work are as follows. I miss the early embryonic cells, the cycles of control points on, ZM has no detectable effect on the two FREQUE