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Fig. 10. Stability of TPPP/p25 bundled microtubules. (A) For the in vivo assay, transfected cells were treated with 100 nM vinblastin in the final 2 hours of the incubation period. Tubulin was detected by immunocytochemistry (red). Green signal originated from EGFP-TPPP/p25. In the presence of vinblastin, in non-transfected cells (red only), the microtubular network is completely dissolved. Fine fibers in the cytoplasm are not detected after vinblastin treatment, but bundled, tubulin-positive fibers were preserved. Note the colocalization pattern of red and green signal in merged image in A. (B-D) Effect of low temperature (4°C; B), plus 0.75 mM Ca2+ (C) or plus 5 µM vinblastin (D) on the taxol-stabilized microtubules treated (
) and untreated (
) with TPPP/p25. 15 µM tubulin was polymerized by addition of 20 µM taxol with and without 3 µM TPPP/p25 at 37°C for 15 minutes in PEM buffer. Ca2+ and vinblastin were added and the depolymerization of microtubules was followed by turbidimetry for 60 minutes at 4°C. Bar, 10 µm.
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