Tgf β

In brief, an equal amount of protein was separated with 10%–12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to 0.22-μm polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Thereafter, the membranes were blocked with 5% milk and immunoblotted with primary antibodies. The antibodies include PTEN (1:1,000; Bioss, Beijing, China), P53 (1:500; Bioss, Beijing, China), TGF-β (1:1000; Bioss, Beijing, China), and GAPDH (1:2000; Zsbio, Beijing, China).

Equal amounts of protein were lysed with equal amounts of precooled lysis buffer containing 10 µL of the serine protease inhibitor phenylmethyl–sulfonyl fluoride per mL of buffer. After being incubated for 45 min on ice, the cell lysates were centrifuged at 12,000 × rpm at 4℃. The supernatant was collected, quantified using BCA (Bicinchoninic Acid) protein assay kits, and mixed with 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis loading buffer. The sample was then boiled for 5 min. Subsequently, equal amounts of protein were separated through 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Germany). The membranes were blocked at room temperature for 2.5 h in 5% nonfat dry milk in TBS-T. The membranes were incubated on a shaker overnight at 4 ℃ with rabbit anti-human VSIG4 (1:1,000, Bioss, China) and TGF-β (1:1,000, Bioss, China); GAPDH (1:40,000, Proteintech, China) was used as a loading control. The membranes were washed six times with TBS-T for 6 min each time and subsequently incubated with the appropriate secondary antibody for 2 h at room temperature. Immune complexes were visualized with an enhanced chemiluminescence detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were determined using Image J software.