A53227

Total protein concentration was extracted from HeLa cells using a RIPA lysate (89901, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a protein detection kit (A53227, Thermo Fisher Scientific, Waltham, MA, USA). A volume of 30 μg protein for each cell was transferred to a PVDF membrane (HVLP04700, Millipore, Billerica, MA, USA) using the SDS-PAGE method. After the transfer was completed, the membrane was soaked from bottom to top with TBS, then placed in an incubation box containing a 5% skimmed milk powder solution (37°C, 1 h), incubated with shaking at room temperature on a decolorization shake flask for 2 hours, and then incubated with phosphorylated (p)-Akt (1: 1000; 56 KD; ab38449; Abcam, Cambridge, UK), Akt (1: 500; 55 KD; ab8805), Bax (1: 1000; 21 KD; ab32503), Bcl-2 (1: 1000; 26 KD; ab59348), Wnt1 (1 μg/mL; 41 KD; ab85060), MMP-9 (1 μg/mL; 95 KD; ab73734), EGFR (1: 1000; 134 KD; ab52894), β-catenin (1: 1000; 92 KD; #9562; Cell signaling technology, Danvers, MA, USA), or β-actin (1: 1000; 45 KD; #4970) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1: 5000; ab205718; Abcam, Cambridge, UK) for 2 h. Finally, the signals were detected using SignalFir ECL reagent (#6883) and the gray value of the band was analyzed and calculated using ImageJ (version 5.0, Bio-Rad, Hercules, CA, USA) [17 (link)].