SAnxA1 binds and activates human FPR2. (A) Analysis of competition binding in FPR2-HEK293 cells using AnxA1 and SAnxA1 to compete with the tracer (W peptide; 82pM). Representative of 2 experiments in triplicate. (B) FPR2-HEK293 cells were incubated with 1nM AnxA1 and SAnxA1, for the indicated times, to determine the level of Erk1/2 phosphorylation by Western blot using anti–p-Erk1/2 antibody. The bar graph reports the densitometric analysis of the p-Erk2 bands shown on the left. Representative of 3 experiments. Calcium mobilization in (C) FPR2-HEK293 cells and (D) isolated PMNs. In all cases, cells were loaded with 2μM Fura-2 and analyzed for [Ca²⁺]i changes after 0.1-1nM AnxA1 or SAnxA1. Fluorescence ratios tracing of representative experiment recorded in a dual excitation fluorescent spectrophotometer are shown (left panels). Maximal intracellular calcium increased induced by different concentration of AnxA1 or SAnxA1 (0.1 or 1nM) were normalized to the response induced by ionomycin (1μM; taken as 100%; right panels). Values are expressed as the mean ± SEM of 3 independent experiments.