AnxA1_Ac2-26 dampens ERK and Akt activation in neutrophils isolated from patients with SCD and activates cleaved caspase-3. (A) Procedure for sample preparation for western blot analysis. Representative western blots of neutrophils from control volunteers and patients with SCD for ERK (B) and Akt (C) activation after AnxA1_Ac2-26 treatment and ionomycin stimulation. Densitometric analysis of p-ERK/total ERK (D; n = 5) and p-Akt/total Akt (E; [n = 4]. One outlier [defined as ≥2 standard deviations] removed from control ionomycin+AnxA1_Ac2-26 [30 minutes]). (F-G) Percentage of H3Cit⁺ SCD neutrophils after stimulation with ionomycin (4 µM, 3 hours), with/without pretreatment with AnxA1_Ac2-26 (30 µM, 15 minutes) and Akt (10 µM, 30 minutes), ERK (10 µM, 60 minutes), or caspase-3 (Z-DEVD-FMK, 20 µM, 45 minutes) inhibitors (n = 5 each group). (H) Representative immunofluorescence images of cleaved caspase-3 staining from SCD neutrophils with/without AnxA1_Ac2-26 (30 μM) treatment. Scale bar, 100 µm. (I) Percentage of cleaved caspase-3⁺ neutrophils from control and patients with SCD with/without AnxA1_Ac2-26 treatment (n = 5). Data expressed as mean ± SEM from independent experiments. *P < .05; **P < .01; ***P < .001 vs unstimulated control neutrophils. ^#P < .05; ^##P < .01 vs unstimulated SCD neutrophils. ^$$P < .01; ^$$$P < .001; ^{$ $ $ $}P < .0001 vs ionomycin-stimulated SCD neutrophils at the corresponding time points. ^ΔΔP < .01 vs AnxA1_Ac2-26-pretreated, ionomycin-stimulated SCD neutrophils.