FIGURE 2.

Serine protease activity is required for RIPK3-dependent MLKL phosphorylation, and subsequent p38 MAPK and PI3K activation in CD44-induced necroptosis in GM-CSF-primed neutrophils. (A–C and E–G) Immunoblotting. Neutrophils were cultured as indicated and subsequently stimulated with anti-CD44 mAb for 5 min. (D) Immunoblotting. Neutrophils were cultured in the presence and absence of compound 1 (40 μM) or compound 3 (20 μM) for 1 h. Cell lysates were analyzed by immunoblotting for phosphorylated MLKL, phosphorylated AKT, and phosphorylated p38 MAPK. MLKL, AKT, p38, and GAPDH expression levels were analyzed as loading controls. Representative immunoblots are shown (n ≥ 3). (A) Upper panel: p-MLKL protein expression levels were quantified relative to the control condition of MLKL. Data are from 3 independent experiments. **p < 0.01, ***p < 0.001.