Protein Expression Analysis by Western Blot

Total protein was isolated from RIPA lysate and quantified using the BCA method. Next, 30 μg of total protein was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk at 37 °C for 2 h, the membrane was incubated with primary antibodies against LDHA (1:1 000), CD47 (1:1 000), PDIA3 (1:1 000), SLC16A1 (1: 200), and the internal reference β-actin (1:1 000) overnight at 4 °C. The next day, the membrane was incubated with secondary antibodies at 37 °C for 1 h. The bands were detected by adding the chemiluminescence substrate, and proteins were quantified by densitometry using Image Lab 5.2 software, with β-actin as the internal reference. The experiment was performed three times.