ΔICL19 mutation blocks FAK activation and cell migration. (A) Whole-cell lysates from stable SNU761 cells expressing FLAG-TM4SF5 WT or ΔICL19 deletion mutant in suspension (Sus) or reseeded on FN as described in the Materials and Methods for the indicated times were immunoprecipitated with anti-FLAG antibody before immunoblotting for the indicated molecules. (B) SNU449 cells transfected with pEGFP-TM4SF5 were wounded and incubated for 6 h, before staining with phalloidin. (C) Transwell migration assays (for 12 h) were performed for cells transfected with mock, FLAG-TM4SF5 WT, ΔICL13, or ΔICL19 deletion mutants. Five random images for the migrated cells were calculated and mean ± standard deviation values for graphic presentation. (D) SNU449Tp cells transfected with mock, FLAG-TM4SF5 ΔICL13, or ΔICL19 deletion mutants for 48 h were harvested before standard western blotting for the indicated molecules. (E) Chemotaxis or haptotaxis (for 12 h or 16 h, respectively) of SNU761 cells stably transfected with FLAG-TM4SF5 WT, ΔICL13, or ΔICL19, compared with control migration toward 1% BSA, as explained in the Materials and Methods. *P≤0.05 against mock control; **P≤0.05 against TM4SF5 WT cells. Data represent three independent experiments.