Figure 2
Purification and activity of epitope-tagged MHCK-C. A. MHCK-C expression levels are indicated by western blot analysis of total cell lysates of the 3xALA parental cell line (3XALA lane) and lysates of 3xALA cell overexpressing FLAG-MHCK-C (3x/FLAG-MHCK-C lane). Immunoreactivity of purified FLAG-MHCK-C indicates presence of full length and clipped FLAG-MHCK-C (pure FLAG-MHCK-C lane). Coomassie blue stained material (Coomassie lane) indicates purity and the presence of a clipped breakdown catalytic domain fragment migrating at ~ 35 kDa. Western blot performed with polyclonal antisera generated against the catalytic domain of MHCK-C. B. FLAG-MHCK-C both autophosphorylates and phosphorylates Dictyostelium myosin II on the heavy chain. C. Kinetics and stoichiometry of myosin heavy chain (MHC) phosphorylation by FLAG-MHCK-C. For panels B and C phosphorylation was performed in a reaction mixture consisting of 500 nM MHC (in the form of native myosin II), 100 nM FLAG-MHCK-C, 0.5 mM ATP, 2 mM MgCl2, and 20 mM TES pH 7.0. Error bars represent S.E.M., n = 3