Fig. 6.

Immunoprecipitated HA-Pfcrk-3 associates with protein kinase and histone deacetylase activities. (A) Association of HA-Pfcrk-3 with kinase activity. Immunoprecipitates obtained with anti-HA antibodies from untransfected wild-type 3D7 parasites (lane 1) or from parasites expressing HA-tagged Pfcrk-3 (lane 2) were subjected to a kinase activity assay in a standard 30-μl reaction mixture containing 5 μg histone H1 as a substrate. The kinase reactions were then run on a 12% polyacrylamide gel, which was dried and subjected to autoradiography. (B) Histone deacetylase assay. Immunoprecipitates obtained with anti-HA antibodies from untransfected wild-type 3D7 parasites (bar 1) or from the HA-tagged Pfcrk3-expressing cell line (bars 2 to 6) were subjected to HDAC assays (Millipore). With the exception of the experiment for which results are shown in bar 2, the immunoprecipitates were incubated for 16 h at 37°C in a substrate buffer containing the sirtuin cofactor NAD. HDAC inhibitors (10 μM nicotinamide [Nmide] or 2 μM trichostatin A [TrichoA]) were used separately (bars 4 and 6) or together (bar 5). Reactions were terminated with activator solution followed by fluorescence measurements (excitation wavelength, 360 nm; emission wavelength, 460 nm). Error bars represent standard deviations from duplicate experiments.