Fig. 7
From: Activation of STAT3 signaling is mediated by TFF1 silencing in gastric neoplasia
TFF1 negatively regulates IL6-induced STAT3 activation through GP130-IL6Rα axis. a Western blot analysis using AGS cell lines infected with control or TFF1 adenovirus. After stimulation with IL6 (100 ng mL−1) for 30 min, TFF1 expressing cells showed a significant decrease of p-STAT3 (Y705), p-GP130 (S782), and p-JAK2 (Y1007/Y1008) protein levels as compared to control cells. b The relative intensity ratio of p-JAK2/β-Actin, p-GP130/β-Actin, and p-STAT3/β-Actin were calculated by Image-lab software from BioRad. The results are expressed as mean ± SEM of at least three independent experiments. ***P < 0.001 by two-tailed Student’s t test. c Immunoprecipitation and Western blot analysis following IL6Rα pulldown using AGS cells infected with TFF1 or control adenoviruses (5 MOI), with or without treatment with IL6 (100 ng mL−1) for 30 min. The first lane exhibits AGS control following immunoprecipitation with mouse IgG control antibody. All immunoprecipitations and their corresponding input samples were subjected to immunoblotting with rabbit polyclonal antibody against GP130 and IL6Rα. The expression of TFF1 and equal amounts of protein loading were confirmed in the input samples. d Punctate proximity ligation assay (SOURCE) was performed in accordance with supplier’s instructions in AGS cells infected with control or TFF1 adenovirus. The presence of red signals indicates positive ligation, indicative of interaction. Using IL6Rα and GP130 antibodies, the results indicated the presence of IL6Rα–GP130 interaction (red signals), following stimulation with IL6 in control cells (Ctrl). This interaction was not detected in AGS cells expressing TFF1 (upper left two panels). The lower panel displays immunofluorescence following the use of TFF1 and IL6Rα antibodies and demonstrates an interaction between TFF1 and IL6Rα (red signals), (lower right two panels). As a negative control for PLA background reaction, control cells were stimulated with IL6 and probed with a single antibody for GP130 (right single panel). Maximum intensity projection is presented in the upper and right side of each image
