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. 2018 Feb 28;19(3):684. doi: 10.3390/ijms19030684

Figure 2.

Figure 2

FASL induces apoptosis in neutrophils, which switches to necroptosis when caspases are inhibited. (a) WT and Bid−/− neutrophils were treated with FASL (100 ng/mL) for 0–8 h. Lysates were assessed by immunoblotting. Presented immunoblots are representative of at least two independent experiments. * indicates non-specific bands; ** indicates bands derived from previous anti-BID immunoblotting. (b) WT and Bid−/− neutrophils were stimulated with FASL (100 ng/mL) for indicated time points. Lysates were assayed for caspase-3/-7 activity by fluorogenic DEVDase assay. n ≥ 3, mean ± SEM. p < 0.05 (*), p < 0.01 (**). (c) WT and Bid−/− neutrophils were pre-treated with Q-VD-OPh (20 μM) for 30 min and subsequently treated with FASL (100 ng/mL) for 6 h. Lysates were subjected to immunoblotting. Presented immunoblots are representative of at least two independent experiments. (d) WT and Bid−/− neutrophils were pre-stimulated with Q-VD-OPh (20 μM) for 30 min followed by administration of FASL (100 ng/mL) for 16 h. Fractionation by phase separation was performed and lysates were subjected to immunoblotting. Presented immunoblots are representative of at least two independent experiments. All experiments were performed with in vitro differentiated neutrophils.