Fig. 3. Identification of phosphorylation sites in AtMAP65-1. (A) Phosphorylation of four AtMAP65-1 fragments (FR1-4) using the M-phase extract (M-phase) or with the microtubule-associated proteins preparation (MTP). Cell extract without substrate was used as a negative control (Kinase). CBB, Coomassie Brilliant Blue R-250 staining; Autorad, autoradiogram. (B) Phosphorylation of synthetic peptides (PA-PI) containing putative phosphorylation sites in Fragment 4 using the M-phase extract. The inset shows the total counts per minute (CPM) in the corresponding reaction mixtures. (C) Autoradiogram (Autorad) of the recombinant wild-type AtMAP65-1 (WT) and the mutant, AtMAP65-1^9D (9D) phosphorylated with M-phase or S-phase (interphase, sampling point 0) extracts. The corresponding nitrocellulose membrane was stained with amido-black (Amidoblack).