FIG. 4.

Association of Smad1 and mZnf8 in mammalian cells. (A) A total of 0.2 μg of pFA-CMV (GAL4DB [GDB] vector alone) or GAL4DB-Smad1 was cotransfected with various amounts of Znf8-vp16 (as indicated in the figure) as well as the reporter construct (Gal)5-E1b-lux into HEK293 cells. Luciferase activities were measured as described in Materials and Methods. The luciferase activity of cells with GAL4DB alone was defined as 10. Znf8-vp16 dramatically increased the reporter activity in cells transfected with GAL4DB-Smad1 in a dose-dependent fashion but had subtle effects on that of cells with the GAL4DB vector alone. Data were averaged from three independent cultures, with error bars indicating standard deviations. (B) mZnf8 fused with GST in a mammalian expression vector was cotransfected into COS-M6 cells with a plasmid expressing HA-tagged Smad1 or HA-tagged domains of Smad1. ALK6 (Q203D), encoding constitutively active BMPRI, was also cotransfected into COS cells in all experiments except for lane 9. The GST (negative control) and GST-mZnf8 fusion proteins were purified with GST beads (see Materials and Methods). The samples were divided into two equal parts. The first half was loaded onto an SDS-PAGE gel, and an HA antibody was applied to detect whether HA-tagged proteins copurified with GST-mZnf8 from cell lysates in Western analysis (top panel). The same filter was stripped and Western analysis with an anti-GST antibody was performed to confirm the precipitation of GST or GST-mZnf8 fusion proteins (middle panel). Expression of HA fusion proteins in COS cells was confirmed by Western analysis with an anti-HA antibody (bottom panel). (C) The left-half samples from lane 1 and lane 2 of panel A were loaded on another SDS-PAGE gel. Western analysis with anti-phospho-Smad1 antibody was performed to test whether the phosphorylated Smad1 (active form) copurified with GST-mZnf8.