FIGURE 1.

SETD6 interacts and methylates PAK4 in vitro. A, protoarray experiment showed a positive signal for methylation of recombinant PAK4 protein by GST-SETD6, contrary to GST protein as a negative control (13). Protoarrays were incubated overnight under PKMT reaction conditions with GST-SETD6 and GST as a negative control. Arrays were then probed with a pan-methyl antibody followed by a fluorophore-conjugated secondary antibody. B, in vitro methylation assay in the presence of ³H-labeled SAM with recombinant His-Sumo-PAK4, BSA (negative control), and GST-SETD6. Coomassie stain of the recombinant proteins used in the reactions is shown on the bottom. C, HEK-293T cells were transfected with FLAG-PAK4 or empty plasmid. Cell extracts were immunoprecipitated (IP) with FLAG M2 beads, followed by a radioactive in vitro methylation assay in the presence of GST-SETD6. Coomassie Blue stain shows the immunoprecipitated FLAG-PAK4 and the added GST-SETD6. Anti-FLAG Western blot represents input of FLAG-PAK4 transfected to HEK-293T cells (bottom panel). D, ELISA-based analysis of the interaction between recombinant GST-SETD6 and His-PAK4. The 96-well plate was coated with 2 μg of His-PAK4 or BSA as control, and then covered with 0.5 μg of GST-SETD6 or GST. Signal detection was achieved using primary anti-GST antibody and secondary HRP-conjugated anti-rabbit antibody and normalized to GST signal. Data are from at least three experiments (error bars, S.E.).