NCI-H295R cells and APA tissues were collected, lysed with lysate (containing protease inhibitors), and seeded in culture plates for cell culture. The proteins were then collected and separated by electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. After washing the membrane, 5% skim milk was added and it was sealed at room temperature for 1–2 h. The membrane was then placed in a primary antibody diluent and incubated overnight at 4 ℃. Next, the membranes were washed with a tris buffered saline tween (TBST) solution and added with HRP-labeled goat anti-rabbit IgG diluent (1:10,000, Solarbio) and incubated at room temperature for 2 h. After washing the membrane, the enhanced chemiluminescence (ECL) chemiluminescence method (Solarbio life science, Beijing) was utilized to detect the bands on the membrane. Image J software (National Institutes of Health, USA) was used to determine the absorbance value of the protein bands.
The primary antibodies used in the experiment included the following: rabbit anti-Sfrp2 antibody (1:500, Beyotime, Beijing), rabbit anti-Wnt5a (1:600, Beyotime, Shanghai), rabbit anti-LEF (1:1,000, Abcam), rabbit anti-TCF (1:1,000, Abcam), and rabbit anti-CYP11B2 (1:2,000, Abcam).
The primary antibodies used in the experiment included the following: rabbit anti-Sfrp2 antibody (1:500, Beyotime, Beijing), rabbit anti-Wnt5a (1:600, Beyotime, Shanghai), rabbit anti-LEF (1:1,000, Abcam), rabbit anti-TCF (1:1,000, Abcam), and rabbit anti-CYP11B2 (1:2,000, Abcam).