Figure 7.

Interaction between Smad7, p38, and MKK3/6 in vivo. (A) Smad7 binds to p38 in vivo. COS1 cells were transfected with Myc-tagged Smad7 alone or together with wild-type (WT) or DN Flag-tagged p38, with or without constitutively active TGF-β type I receptor (c.a. ALK-5). Lysates were immunoprecipitated with anti-Flag antibodies and the immunoblots were incubated with antibodies specific for Myc or Flag. (B) Endogenous Smad7 binds to phosphorylated p38 in vivo. PC-3U cells were treated or not with TGF-β1 for the indicated time periods. Cell lysates were immunoprecipitated with anti-Smad7 antibodies and the immunoblots were incubated with antibodies specific for phospho-p38. Immunoblots from total cell lysates were incubated with antibodies specific for Smad7, phosphorylated p38, and p38. Smad7 binds to p38 in vivo. (C) COS1 cells were transfected with Flag-tagged Smad7 alone or together with wild-type HA MKK3 or HA-MKK6. Lysates were immunoprecipitated with anti-Flag antibodies and the immunoblots were probed with antibodies specific for HA. Immunoblots from total cell lysates were incubated with antibodies specific for HA and Flag. A clear MKK3 band is seen; upon prolonged exposure, a faint, specific MKK6 band is also seen.