Anti e cadherin

Total protein was lysed from cells using radioimmunoprecipitation assay buffer (RIPA buffer; Beyotime, Shanghai, China). Protein quantification and western blotting assays were performed as described previously [21 (link)]. The following primary antibodies were used: anti-E-cadherin (WL01482, Wanleibio, China), anti-ETS1 (#14069, Cell Signaling Technology (CST), USA), anti-N-cadherin (WL01047, Wanleibio, China), anti-Snail (#3879, CST), anti-EIF4A3 (ab18057, Abcam, USA), and anti-Vimentin (ab8069, Abcam).

Total protein was extracted using a cell lysis buffer for Western blotting and immunoprecipitation (Beyotime, Shanghai, China) containing PMSF and a protease inhibitor cocktail. Proteins (30 μg/well) were separated using 10% SDS/PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies used in this study were against anti-HAPSTR1 (1:1000, OTl2B8; Origene), anti-HAPSTR1 (1:500, OTI2D1; Novusbio, Centennial, CO, USA), anti-LRPPRC (1:5000, 21175-1-AP; Proteintech), anti-β-actin (1:1000, 20536-1-AP; Proteintech), anti-Ub (1:1000, 10201-2-AP; Proteintech), anti-N-cadherin (1:5000, 66219-1-Ig; Proteintech), anti-Beclin1 (1:2000, T55092; Abmart, Berkeley Heights, NJ, USA), anti-P62/SQSTM1 (1:2000, T55546; Abmart), anti-ATG5 (1:2000, T55766; Abmart), anti-vimentin (1:500, WL01960; Wanleibio), anti-snail (1:500, WL01863; Wanleibio), and anti-E-Cadherin (1:500, WL01482; Wanleibio).

Western blot analysis was performed using an extraction buffer as described [44 (link)]. MKN-45, MKN-28 and U87 cells were cultured in serum-free medium overnight, then treated with the indicated dose of peptide CM 7 for 24 h at 37 °C after stimulated without or with HGF for 15 min. Next, total cell lysates were separated by 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with following primary antibodies: anti-phospho-Met (Tyr1234/1235, (Cell Signaling Technology, Beverly, MA, USA), anti-Met (Affinity, USA), anti-phospho-AKT (Ser473, (Affinity, USA)), anti-AKT (Affinity, USA), anti-phospho-Erk1/2 (Thr202/Tyr204, (Affinity, USA)), anti-Erk1/2 (Affinity, USA), anti-E-cadherin (WanleiBio, Shengyang, China), and anti-GAPDH (Multi Sciences, Hangzhou, China). Then, horseradish-peroxidase-conjugated anti-rabbit IgG was used as a second antibody. The immunoreactive proteins were exposed using an enhanced chemiluminescence detection reagent (WanleiBio, Shengyang, China). After sacrificing the mice and isolating the tumor tissue, Western blot was performed as previously described.