Fig. 3

ALK/Akt/NF-κB axis in Em Ca cells. a Left: Hec251 cells stably overexpressing full-length ALK (H251-ALK#8 and #16, two independent clones of stable cells). Western blot assay detected exogenous ALK protein (220 kDa) and a second protein of approximately 150 kDa (left). Right: note the strong cytoplasmic ALK staining in the two independent stable cell lines. b Left: phase-contrast images of Hec251 cells stably overexpressing full-length ALK (H251-ALK#16) and the mock cells treated with 2.5 ng/ml TGF-β1 or 50 ng/ml HGF. Right: western blot analysis for the indicated proteins in ALK#16 and mock cells. con, control; T, TGF-β1; H, HGF; N, nuclear c Western blot analysis for the indicated proteins in Ishikawa cells after cotransfection of ALK and shRNAs against ALK. N, nuclear d Analysis of the indicated protein expression levels by western blot assay in Ishikawa cells after treatment with 20 ng/ml TNF-α for the time shown. N, nuclear e Left: analysis of mRNA levels for the Snail, Slug, and Twist1 genes by real time RT-PCR in Ishikawa cells following transfection of p65. Middle: analysis of the indicated protein expression by western blot assay in Ishikawa cells after transfection of p65. Exogenous p65 is indicated by an asterisk in the p65 panel. con, control. Right: Ishikawa cells were transfected with Snail, Slug, and Twist1 reporter constructs, together with p65. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate