Figure 2.

ErbB1 and ERK1/2 are active in Wnt-HC11 cells. (A, B) C-HC11 and Wnt-HC11 cells were either incubated for 10 min with 100 ng ml⁻¹ EGF and/or for 1 h with 5 μM PKI166, or left untreated before preparing whole cell lysates. (A) ErbB1 was immunoprecipitated from 500 μg whole cell lysate and immunoblotted with a phosphotyrosine specific antibody (P-ERK indicates tyrosine-phosphorylated ERK); membranes were stripped and reprobed for ErbB1. (B–E) Samples of whole cell lysate (50 μg) were immunoblotted with a phospho-ERK1/2 antibody; membranes were stripped and reprobed for ERK1/2 or ERK1. (C) C-HC11 cells were incubated for 10 min with conditioned media from C-HC11 and Wnt-HC11 cells; where indicated, cultures were pretreated for 1 h with PKI166. (D) Conditioned media from untransfected (control) or sFRP1-expressing 293 cells was premixed for 1 h with an equal volume of conditioned media from C-HC11 or Wnt-expressing HC11 cells. These mixtures were then added to C-HC11 cultures for 10 min; where indicated, cells were incubated with EGF for 10 min. (E) C-HC11 cells were pretreated 1 h with conditioned media from untransfected or Dkk1-expressing 293 cells, followed by incubation of cultures with conditioned media from C-HC11 or Wnt-HC11 cells for 10 min.