Bl615a

MDA-MB-231 and MCF-7 cells were inoculated in 6-well plates at the density of 5.0×10 ^5 cells/well and treated with Syringin (0, 160, 320 µg/mL) for 48 h. Then, the RIPA lysis kit (BL504A, Biosharp, Anhui, CHINA) was mixed with PMSF (BL504A, Biosharp, Anhui, CHINA) and PI (BL615A, Biosharp, Anhui, CHINA) for cell lysis. After 12000 rpm centrifugation for 10 min, the total proteins of the whole cell lysate were quanti ed by a BCA protein assay kit (69107317, Biosharp, Anhui, CHINA). The same total proteins were separated on 10% SDS-PAGE gel electrophoresis (AS1012, ASPEN, CHINA) and then electrophoretically transferred to a nitrocellulose membrane by electrophoresis (DYY-6C, Beijing, CHINA). After sealing with 5% BSA at room temperature for 1.0 h, the membranes were washed 3 times and incubated with 13 primary antibodies respectively (including p-EGFR, PIK3CA, p-PIK3CA, etc.) at 4°C overnight. The next day, washed the membranes with tris buffered saline + Tween (TBST) 5 times for 30 minutes. Fresh ECL (170-5060, Bio-Rad, USA) mixed solution was added to the protein side of the membranes and detected by luminescence. The lm was scanned and archived, and the optical density of the band was analyzed by the AlphaEaseFC (Alpha Innotech, USA).