Western Blot Assay

The radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to lyse the treated bEnd.3 brain endothelial cells for 15 min on ice and the proteins were quantified using a bicinchoninic acid (BCA) protein quantitative kit (Beyotime, Shanghai, China). Subsequently, approximately 30 μg samples were loaded and separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then transferred to a poly(vinylidene difluoride) (PVDF) membrane, followed by blotting with antibodies against KLF6 (1:1000, Cat#sc-365633, Santa Cruz Biotechnology) and COX-2 (1:1000, Cat#12282, Cell Signaling Technologies) for 2 h at room temperature. After washing with Tris-buffered saline with Tween 20 (TBST) buffer, the membranes were incubated with the secondary anti-rabbit (1:1000, #7074, Cell Signaling Technologies) and anti-mouse antibodies (1:1000, #7076, Cell Signaling Technologies) for 1.5 h at room temperature. β-Actin (1:10 000, Cat#4970, Cell Signaling Technologies) was used as the negative control. Images were taken and analyzed with the software Image J.