Quantitative Protein Analysis via Western Blot

Proteins were extracted with RIPA buffer (Beyotime Biotechnology, China)) containing protease and phosphatase inhibitors (Beyotime Biotechnology, China)) and were quantified using Rapid BCA Protein Assay Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. Western blot analysis was performed as previously described^{19 (link)}. Briefly, 40 μg lysate was loaded onto SDS-PAGE gels, blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), and incubated with antibodies. The primary antibodies used in this work including anti-IRF4 (Proteintech, 11247-2-AP, dilution 1:1000), anti-FSTL1 (Abcam, ab71548, dilution 1:1000), anti-β-Tubulin (Abclonal, AC008, dilution 1:5000), anti-GAPDH (Abways, AB0037, dilution 1:5000), anti-flag (Abclonal, AE005, dilution 1:2000).