Protein Expression Analysis by Western Blotting

The protein expression levels of model-related genes were verified by Western blotting. Samples were treated with RIPA lysis buffer. A BCA assay kit (Thermofisher, USA) was used to determine the protein concentration. An equal amount of protein (20 μg) was loaded into lanes. After being separated by electrophoresis, proteins were electrically transferred to a PVDF membrane (Millipore, USA). After blockaded with 5% milk, the membrane was incubated with the primary antibodies: anti-PDIA4 (SAB1404743, Sigma-Aldrich, St. Louis, MO, USA), anti-DUSP6 (SAB1410312, Sigma-Aldrich, St. Louis, MO, USA), anti-PTPRN (ab207750, Abcam, Cambridge, MA, USA), anti-PILRB (ab198267, Abcam, Cambridge, MA, USA), anti-CBLN1 (ab181379, Abcam, Cambridge, MA, USA), and anti-GAPDH (ab181602, Abcam, Cambridge, MA, USA), each was diluted at a ratio of 1:1000 and incubated over-night at 4 °C. After incubation with the corresponding horseradish peroxidase-linked secondary antibody at room temperature for 1 h, target proteins were developed by the enhanced chemiluminescence kit (Millipore, USA).