Figure 3.

Binding of BT173 to HIPK2 disrupts its PPI with Smad3. (A) DARTS confirms a direct binding of BT173 to HIPK2. Cleared cell lysate was preincubated with 100 μm BT173 or DMSO vehicle for 1 hour. Lysates were then digested with varying dilutions of pronase for 20 minutes and immunoblotted for HIPK2 and GAPDH loading control. Undigested lysate was used as a positive control. (B) Cell lysates were preincubated with varying concentrations of BT173 and digested with pronase (1:10,000 dilution) as in above experiment. Increasing concentration of BT173 results in greater protection of HIPK2 from pronase digestion. (C) The 293T cells expressing His₆-HIPK2 were incubated with DMSO vehicle or BT173 (3.3 or 10 μM) for 16 hours. His₆-HIPK2 was pulled down with cobalt beads, and eluted protein complexes were subject to immunoblotting. Total cell lysates were immunoblotted with indicated antibodies as controls.