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Fig. 8. Cyclin B and Msps are not destabilised in the absence of Hsp90, and cyclin B can interact directly with MTs in vitro. (A) Western blot of wild-type and Hsp90 neuroblasts. The brains were dissected and either incubated at 25°C for 2 hours or heat shocked at 37°C for the same amount of time. The filters were probed with anti-Msps, anti-cyclin B and anti-Polo antibodies. CP190 was used as loading control. In Hsp90 mutants, Polo protein is not stabilised (see de Carcer et al., 2001) but levels of Msps and cyclin B are comparable to, or even higher than, levels in WT. (B) Cyclin B can associate directly with MTs in vitro. Western-blot of a MT co-pelleting assay developed with affinity purified MBP antibodies. Supernatants (S) and pellets (P) from a control sedimentation assay with no MTs (left) or with MTs (right) performed with the same amount of the following proteins (top to bottom) MBP; MBP-CBFL, MBP-CBNT, MBP-CBC2. When MTs are not present all the proteins are detected in the (S) fraction. When incubated with MTs, MBP is still detected in the S fraction but a shift in MBP-CBFL and MBP-CBNT to the P fraction is detected. MBP-CBCT1 seems to be equally distributed into S and P fractions, suggesting that the N-terminal domain of cyclin B is required for MT binding.
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