Fig. 3. Binding of EB1 and the C-terminal EB1 domain to MTs or APC in vitro. (a) MT pelleting assay. Lanes 1-3 show 1/20 of the respective MBP fusion proteins after preclearance by centrifugation and before incubation with polymerized tubulin. Lanes 4-6 show 2/3 of the MT pellets after incubation with the respective MBP fusion proteins and centrifugation through a glycerol cushion. Lane 7, 2.5 µg purified bovine brain tubulin. (b) Binding of MBP-EB1 fusion proteins to the EB1-binding domain in GST-APCCT. Precipitation of the respective MBP fusion proteins by GST-APCCT bound to APC AB/Protein A (APC AB+GST-APCCT) (lane 7-9). GST-APCCT appears as a double band of which the faster migrating one is probably caused by partial degradation. In control assays, MBP fusion proteins were incubated with anti-GST antibody (GST AB) bound to Protein A (lane 1-3); with GST bound to GST AB/Protein A (GST AB+GST) (lane 4-6) and with anti-APC antibody (APC AB) bound to Protein A (lane 10-12). Approximately 1/15 of the total MBP fusion proteins used for each binding assay are shown in lane 13-15. M, Molecular weight standards.

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