Fig. 7. Proteolysis of tubulins in the crude extract of A. arbuscula revealed by immunoblotting. 75 µg crude extract proteins were mixed with 3 µg CDP II, made up to a total volume of 120 µl with reaction buffer containing 5 mM Ca²⁺ and 10 mM ß-mercaptoethanol and incubated at 37°C. 20 µl samples were withdrawn at intervals, added with 5 µl sample buffer and processed for western blotting as described in Materials and Methods. (A,B) Ponceau-stained proteins on nitrocellulose membrane after SDS-PAGE and electro-transfer. The lanes a-g correspond to the time course reaction with the CDP II as shown below (C,D). (C) Crude soluble protein digest immunoblotted with -tubulin antibodies. (a) 0 minute incubation; (b) 10 minute incubation; (c) 20 minute incubation; (d) 30 minute incubation; (e) 60 minute incubation; (f) soluble proteins without enzyme; (g) soluble proteins + enzyme minus calcium. (D) Same as above but revealed with ß-tubulin antibodies