Ab75478

Lysis buffer (Sigma, USA) was added to the cells to isolate total protein. The protein concentration was determined using a bicinchoninic acid assay protein assay kit. Proteins (25 μg/lane) were separated by SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA). The membrane was blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 h at room temperature, and then probed overnight at 4 °C with the following primary antibodies: anti-IRF6 (1:1000; ab123880; Abcam, Cambridge, MA), anti- KIF20A (1:1000; ab7091; Abcam, Cambridge, MA) (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-GAPDH (ab75478; Abcam, Cambridge, MA). After washing, the blots were incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA). The protein bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the blots were analyzed by Image J software.

To investigate the protein changes related to decreased S100A9 expression in ASMCs, the cells were lysed (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 25 mM NaF, 10 μM ZnCl₂, and protease and phosphatase inhibitor tablets (Roche), pH = 7.5) and incubated on ice for 60 min and centrifuged at 10,000 ×g for 10 min to obtain the cell-free extracts. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed using the following specific primary antibodies: S100A9 (ab75478; Abcam), p-p38 MAPK (Thr180/Tyr182, 4511; CST, USA), p38 MAPK (8690; CST), and β-actin (4967; CST). Horseradish peroxidase- (HRP-) conjugated secondary antibodies were applied, and the membrane was then developed using the ECL detection system.