Supplementary Figure 5: Tyrosine-phosphorylated claudin-11 recruits and activates Src.

(a) Amino acid sequence of claudin-11 (accession code: O75508). The cytosolic tail is highlighted in red. (b) Western blots of Y418-phosphorylated Src and Y530-phosphorylated Src in SAS-pLKO/SAS-shCLDN11. The representative data were from three independent experiments with similar results. (c) GAP activity assay for analyzing the p190RhoGAP activity in HEK293T cells co-transfected with the p190RhoGAP expression vector (pHA-GRLF1) and a vector expression wild-type (WT) or different tyrosine residue-mutated CLDN11, (n = 3 independent experiments). Data represent mean ± S.D. (two-sided Student’s t test). N.S., no significance. (d) Pull down assay/western blots for analyzing the GTP-bound/total RhoA in SAS cells transfected with plasmids expressing wild-type or different tyrosine residue-mutated FLAG-tagged claudin-11. The data was from one experiment. (e) Confirmation of the efficacy of different inhibitors with the western blots of T202/Y204-phosphorylated ERK1/2, and total ERK1/2 in SAS cells treated with different tyrosine kinase inhibitors. The representative data were from two independent experiments with similar results. (f) Immunoprecipitation-western blots for showing the level of tyrosine-phosphorylated claudin-11 in SAS treated with different tyrosine kinase inhibitors following by seeding on the thick collagen for 30 min (cetuximab, 10 μg/ml for 30 min; all other tyrosine kinases inhibitors, 10 μM for 30 min). The representative data were from two independent experiments with similar results. (g) Left: representative snapshots from phase-contrast videos SAS cells treated with DMSO, FAK inhibitor 14 or cetuximab moving on collagen gels. Solid line indicates the position of cell groups at t = 0 h; shading indicates the position of the same cell groups at t = 7 h. Scale bars, 100 μm. Right: directionality of migration presented in rose plot diagrams for SAS cells treated within DMSO, FAK inhibitor 14 or cetuximab (n = 20 cells). The data is representative of two independent experiments with similar results. (h) Western blots of integrin β1, FAK, and Y397-phosphorylated FAK in SAS-pLKO vs. SAS-shSNAI1. The data was from one experiment. Uncropped images of all blots are shown in Supplementary Figure 8. Source data is provided in Supplementary Table 11.