Western blot analysis of activated ERK1/2, STAT3, and AKT-1, and immunoprecipitation and Western blot analysis of activated c-MET in NK4-treated MM cells. (A) HGF-producing KMS11 and KMS34 cells as well as non–HGF-producing KMS27 cells were treated with 200 nM NK4 protein in RPMI containing 4% dialyzed FCS overnight; the cells were then incubated with or without 50 ng/mL HGF at room temperature for 20 minutes. The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with anti–phospho-ERK1/2, anti–phospho-STAT3, and anti–phospho-Ser473 AKT-1 antibodies. Blots were stripped and reprobed with anti-ERK1/2, anti-STAT3, and anti–AKT-1 antibodies. For evaluation of c-MET activation, MM cell lines were also treated with 200 nM NK4 in RPMI containing 4% dialyzed FCS overnight, and then incubated with or without 50 ng/mL HGF at room temperature for 5 minutes. After immunoprecipitation with anti–c-MET antibody, the immunocomplexes were subjected to SDS-PAGE. Immunoblotting was conducted with anti–phospho–c-MET antibody, and then the same filters were stripped and reprobed with anti–c-MET. (B) Densitometric analysis of the activation of c-MET and signal transducers. (C) Evaluation of c-MET activation in primary MM cells obtained from patient 1. Treatment of the MM cells and immunoprecipitation followed by Western blot analysis were performed as described.