Ab169530

We collected 5 ml of peripheral blood from individuals in cohort 2. PBMCs were isolated with density-gradient centrifugation, then washed twice and lysed in RIPA buffer (Sigma-Aldrich, MO, USA) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, MA, USA). Protein concentration of lysates was determined using the BCA Protein Assay Kit according to the manufacturers’ instructions (A53225, Thermo Fisher Scientific, MA, USA). Cell lysates were boiled for 10 min at 95°C with SDS and subjected to 12% SDS–PAGE, and then transferred to nitrocellulose membranes (HATF00010, Millipore). The membranes were blocked and then incubated with anti-ELANE (ab131260, Abcam), anti-CD14 (DF13278, Abcam), anti-S100A11(ab169530, Abcam), anti-PGAM1(DF12693, affinity), anti-SERPINB10(DF9894, affinity), anti-BST2 (DF3846, affinity), and anti-β-actin (AF7018, affinity) overnight at 4°C. The membranes were washed and incubated with anti-rabbit or -mouse IgG-HRP (S0001, affinity) for 1 h. Protein bands were visualized with the western blotting detection system Tanon-5200 (Bio-Tanon, China). Gray value analysis was done by ImageJ (v.1.50g, NIH) software.