Signaling pathways involved in NF-κB activation in HMC-1 cells. (A) Cells were incubated with K-252a (50 ng/mL; ▵), LY294002 (20 μM; □), Ro31-7549 (10 μM; ▴), or PD98059 (20 μM; •) for 24 hours, and cell proliferation was assessed through the trypan blue dye exclusion test. Each point represents the mean ± SE of 6 different experiments. (B) To determine the possible interaction between c-kit signaling and NF-κB activation, we performed EMSA with the use of nuclear extract obtained from cells incubated for 24 hours with different types of signal inhibitors. (C) HMC-1 cells were incubated with various concentrations of IMD-0354 for 24 hours. Spontaneous expression of STAT1, STAT3, STAT5, and STAT6 in HMC-1 cells was detected by Western blotting. Phospho-STAT1, -STAT3, -STAT5, and -STAT6 were determined through immunoprecipitation and Western blotting. Results shown are representative of those of 2 different experiments.