Figure 4.

Reconstitution of p40Met^calpain and evaluation of its apoptotic properties. (a) Equal amounts of extract obtained by lysing MCF-10A cells in LR buffer or (b) 5 ng purified recombinant intracellular Met domain were incubated with the indicated concentration of CaCl₂ and purified calpain 1 for 30 min. (c) Schematic representation of the caspase and calpain cleavage sites in the juxtamembrane domain of Met. (d) MCF-10 A cells were grown for 24 h and either serum starved and treated for 1 h with 1 μM ionomycin (iono) or transfected with a vector expressing the Met S1037 variant. (a, b and d) Cell lysates were analyzed by western blotting with an antibody directed against the kinase domain of human Met. Arrows indicate the positions of Met, recombinant Met, and p40Met^calpain. (e) HEK cells were transfected with empty vector or a vector expressing Flag-tagged versions of p40Met^caspase, p40Met^caspase K1108A (kinase dead, KD), or p40Met^calpain. Twenty-four hours later, the cells were lysed and lysates were analyzed by western blotting with an antibody directed against Flag. (f) MCF-10A cells were cultured on glass coverslips and transfected with the indicated Flag-p40Met. Twenty-four hours later, the cells were fixed and stained with antibodies against Flag and active caspase 3. Flag-positive cells (expressing p40 Met) and displaying active caspase 3 staining were counted. About 1000 flag-positive cells were counted per well and percentage was calculated (n=3; ±S.D.). The P-value of Student's t-test is shown (*P<0.05)