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Fig. 2. CAP350 binds microtubules through an N-terminal domain and not through the CAP-Gly domain. (A) HeLa cell lysate in which tubulin was added or not (see Materials and Methods) and treated with either Nocodazole (NZ) to prevent tubulin polymerisation or with Taxol to stabilise the microtubules. After a 1-hour incubation the cell lysate was centrifuged through a glycerol cushion. Proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE and western blotting using anti-tubulin, anti-p150 and anti-CAP350 antibodies. Note that a small amount of CAP350 and p150 specifically co-precipitated with microtubules in the absence of exogenous tubulin, which significantly increased after addition of tubulin in the lysate. (B) Different partial constructs of CAP350 as indicated on the figure were transcribed in reticulocyte lysate and analysed for their binding to pre-formed microtubules (+MTs). Control experiments were performed without pre-formed microtubules (–MTs). As in A, proteins from the supernatants (S) and pellets (P) were analysed by SDS-PAGE and exposed for different times using a phosphorimager. Only the N-terminal construct (1-983) sedimented with microtubules. This ability was observed with the two N-terminal constructs (C). Arrows indicate the mobility of the in vitro labelled CAP350 constructs.
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