Fig. 5.

Impact of PLEKHA7 on E-cadherin-mediated EGFR signaling activation. a IP performed with anti-EGFR Ab on lysates from OAW42 cells transiently transfected with an empty LZRS (−) or with a LZRS- PLEKHA7 vector (+). Normal rabbit (IgG) serum was used as negative control. Immunoprecipitated samples were analyzed by western blotting with Abs against the proteins reported on the left. The inset above indicates the Myc-tag overexpression corresponding to PLEKHA7 expression. b Left panel: western blotting on total cell lysates from starved empty LZRS vector (−) or LZRS-PLEKHA7 infected OAW42 cells (+) starved and then stimulated with EGF 20 ng/ml for 30 min. Right panel: graph reporting the amount of P-EGFR anf EGFR in EGF stimulated cells evaluated in three different immunoblottings. c Proliferation assay of empty LZRS vector or PLEKHA7 infected OAW42 cells. Asterisks indicate statistically significant values evaluated with one-way Anova. d Left: representative phase contrast images of infected OAW42 cells grown in soft agar for 10 days. Three replicates for each condition were done. Right panel: graph reporting the number of clones/well. Asterisk indicates significant values (p˂0.05). e Left panel: IP with anti-PLEKHA7 Ab on lysates from LZRS vector (−) or LZRS-PLEKHA7 (+) OAW42 cells. Immunoblottings were performed with Abs against the proteins reported on the left. Right panel: quantitative analysis of E-cadherin immunoprecipitated with PLEKHA7 Ab or present in the unbound fraction evaluated in three different experiments as the ratio between the immunoprecipitated (IP) or not immunoprecipitated (Unbound) and the total input. Asterisks indicate significant values (p˂0.001). f Confocal IF performed on infected OAW42 cells immunostained with E-cadherin (E-cadh) together with PLEKHA7, or -β-catenin (cat) Abs. The panel reports the 0.5 μm stacks acquired from the bottom to the top of the cells. Merge images are shown. Bar, 20 μm for xy images; bar, 3 μm for xz images