Itga3

The cellular expression of the six candidate proteins were verified experimentally via Western Blot analysis. Total cell lysates were obtained using RIPA buffer (Thermo Fisher Scientific, United States). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, United States). The membranes were blocked in PBS, 10% (w/v) skim milk for 1 h in phosphate buffer saline-Tween 20 (PBS-T), and incubated for 3 h at RT in 5% milk in PBS-T with primary antibodies: CDH2, EGFR, ITGA3, ITGA5, ITGB1, and CALR (Abcam, Cambridge, United Kingdom). Then, after washing, the PVDF membrane was incubated with secondary antibody (The Jackson Laboratory, United States) for 40 min at RT. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, United States) was used for visualization.

Whole cell lysates were prepared by lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton-X 100, 1mM MgCl₂, MnCl₂ and CaCl₂, 1mM PMSF and 10mM sodium fluoride) [49 (link)]. The primary antibodies used included the following: DPT (HuaAn Biotechnology, Hangzhou, China), focal adhesion kinase (FAK) (Abcam, Cambridge, UK), p-FAK Tyr397 (Abcam, Cambridge, UK), Src (Cell Signaling Technology, Boston, MA), p-Src Tyr527 (Cell Signaling Technology, Boston, MA), ITGA3 (Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Chicago IL). After incubating with the IRDye 680 anti-mouse (LI-COR, Lincoln, NE) and IRDye 800 anti-rabbit (LI-COR, Lincoln, NE) secondary antibodies for 1 hour at room temperature, the bands were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE). Quantification was analyzed by using Image J software.