Characteristics of KATO III/wt-HB-EGF and KATO III/HB-EGF-mC cells. A) Western blot analysis of proHB-EGF and HB-EGF-CTF expression in 3 gastric cancer cell lines. Anti-HB-EGF antibody was used to recognize the proHB-EGF ectodomain and anti-HB-EGF-C antibody was used to recognize the cytoplasmic region of proHB-EGF. Each lane contains 100 μg of protein. Cleavage of proHB-EGF was stimulated using 200 nM TPA. B) HB-EGF-C localization after TPA-inducible processing of proHB-EGF in KATO III/wt-HB-EGF and KATO III/HB-EGF-mC cells by immunofluorescence microscopy. Nuclei were stained blue with DAPI, and HB-EGF-C were stained red with anti-HB-EGF-C antibody. Cells were stimulated with 200 nM TPA for 60 min. C) Western blot analysis of EGFR phosphorylation induced by TPA (200 nM) with or without cetuximab (10 μg/ml) in KATO III/wt-HB-EGF and KATO III/HB-EGF-mC.