FIG 4.

Transgenic CK1.2 is sensitive to D4476. (A) One microgram of recombinant LmaCK1.2 (lanes 1 and 1′), 50 μg of promastigote total protein extract (lanes 2 and 2′), and 50 μg of axenic amastigote total protein extract (lanes 3 and 3′) were analyzed by Western blotting using SY3535 (lanes 1 to 3) or an anti-HsCK1 antibody (lanes 1′ to 3′). (B) Twenty micrograms of extracts obtained from transgenic promastigotes or axenic amastigotes expressing either pLEXSY (mock transfection) or pLEXSY-CK1.2 was analyzed by Western blotting using SY3535 (top) and stained by colloidal Coomassie to evaluate the loading (bottom). (C) As described for panel B, total extracts or immunopurified extracts were analyzed by Western blotting using an anti-V5 antibody (top and middle) and stained by colloidal Coomassie to evaluate the loading (bottom). Numbers to the left of the gels in panels B and C are molecular masses (in kilodaltons). (D) Kinase assay using 27 μM CK-S and 10 μl of V5-coated beads purified from transgenic promastigotes or axenic amastigotes expressing either pLEXSY (mock transfection) or pLEXSY-CK1.2 with or without 10 μM D4476 in 1% DMSO. The activities in the reaction mixtures were measured using a scintillation counter.