FIGURE 5.

SETD6 stabilizes the interaction of PAK4 and β-catenin at chromatin. A, HEK-293T cells were transfected with FLAG-β-catenin alone, with HA-SETD6, or with HA-SETD6 and HA-PAK4. Cell extracts were immunoprecipitated with FLAG M2 beads, and proteins in IP and input samples were detected by Western blot. B, 96-well plate was coated with 3 layers: first with 2 μg of His-β-catenin (white triangle), blocked with 3% BSA in PBST; then 3 μg of His-Sumo-PAK4 or His-Sumo (light and dark circle, respectively); and finally with 0.5 μg of GST-SETD6 or GST. Data are from at least three experiments (error bars, S.E.). C, HEK-293T CRISPR-SETD6 knock-out cells were transfected with the indicated plasmids. Isolated chromatin was immunoprecipitated with FLAG M2 beads, followed by Western blot analysis. Input samples of the chromatin fraction are also shown. D, chromatin fraction derived from wild-type and the CRISPR-SETD6 knock-out MDA-MB-231 cells were subjected to a Western blot analysis with the indicated antibodies. E, HEK-293T cells were transfected with the indicated plasmids and the methylation of immunoprecipitated FLAG-PAK4 at chromatin was detected with a pan-methyl antibody. The level of the chromatin-associated proteins (input) were detected with the indicated antibodies.