Figure 7.

Evidence for a Smad 2/3:Pur protein complex in transfected COS7 fibroblasts. Nuclear protein extracts prepared from COS7 fibroblasts overexpressing either Sp1 (lanes 1–4) or Smad 2/3/4 (lanes 5–8) were IPed with antibodies specific for Sp1 or Smads (as noted at the bottom of each panel) and processed for Western blot (WB) using antibodies shown on the left. Both Sp1 (IP:Sp1) and Smad (IP:Flg-Smad) immunoprecipitates contained Purα and Purβ but samples processed with the Smad 7 negative control antibody did not (IP:Smad 7). None of the overexpressed proteins exhibited nonspecific binding to the affinity isolation beads (no IP). Protein overexpression was verified by Western blot analysis of nuclear protein extracts as shown in the bottom two panels. The band denoted “ns” was not studied in detail but may represent a covalently modified Pur protein isoform (unpublished data).