Enhanced ecl chemiluminescence detection kit

Total cell lysates were prepared in RIPA buffer supplemented with protease and phosphatase inhibitors, then resolved on 8–15% SDS-PAGE gels and electroblotted onto PVDF membrane. The membranes were blocked with a rapid blocking solution (Beyotime, Wuhan, China) and then incubated in primary antibodies prepared in a blocking buffer Beyotime). The membranes were blocked before incubating with anti-PGR (1:1000, Santa Cruz Biotechnology), anti-KLF15 (1:1000, Santa Cruz Biotechnology), anti-TWIST2 (1:1000, Abcam), anti-E-cadherin (1:1000, Santa Cruz Biotechnology), anti-vimentin (1:1500, Proteintech), and anti-GAPDH (1:2000, Proteintech) antibodies overnight at 4°C. On the second day, membranes were washed with PBST, followed by incubating with TBST for 2 h. Then they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, and blots were developed with an Enhanced ECL chemiluminescence detection kit (Vazyme, Nanjing, China) and imaged using the Tanon 5200 ECL chemiluminescence imaging system. Densitometry was performed using ImageJ.

Protein was extracted by RIPA Lysis buffer (Sangon, Shanghai, China), subjected to SDS-PAGE gel, and transferred to PVDF membranes. The membranes were incubated with anti-p-glycoprotein (anti-p-gp, 1:500, Bioss, Beijing, China), anti-myeloid cell leukemia-1 (anti-MCL-1, 1:1,000, Bioss), anti-FBN1 (1:1,000, Bioss), anti-β-catenin (1:2,000, Bioss), anti-c-Myc (1:1,000, Bioss) or anti-β-actin (1:2,000, Bioss). After incubating with Goat Anti-Rabbit IgG (1:20,000, Bioss), the signals were determined by Enhanced ECL Chemiluminescence Detection Kit (Vazyme, Nanjing, China).