Rabbit anti c myc antibody

RIPA lysis buffer (Beyotime, Shanghai, China) was used for protein extraction, and the BCA Protein Assay Kit was used to measure the amount of protein (Solarbio, Beijing, China). Total proteins were then subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). Nonspecific binding sites were blocked using 5% nonfat milk for 2 h and the membranes were incubated at 4 °C overnight with rabbit anti-Wnt2 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-c-myc antibody (1:1000; Bioss, Beijing, China), rabbit anti-CD44 antibody (1:1000; Affinity, Melbourne, United States), rabbit anti-cyclin D1 antibody (1:1000; Abcam, Cambridge, United Kingdom), and rabbit anti-β-tubulin antibody (1:4000; Proteintech, Chicago, United States). The PVDF membranes were then treated for 1 hour with a goat anti-rabbit secondary antibody that was HRP conjugated (1:5000; Bioss, Beijing, China). Enhanced chemiluminescence reagents (Beyotime, Shanghai, China) were used to visualize the bands. Image J was utilized to quantify the chemiluminescent signals of protein bands using β-tubulin as an internal control.