In vitro biologic activity of citrullinated CXCL10 in CHO-CXCR3 cells. (A) The chemotactic activity of CXCL10 and CXCL10-Cit₅ for CHO-CXCR3 cells was measured using a Boyden microchamber (5 or more independent experiments). (B) The amount of phosphorylated ERK1/2 or PKB/AKT (pg/mg total protein) was measured by specific ELISAs after stimulation of serum-starved CHO-CXCR3 cells for 5 minutes with CXCL10, CXCL10-Cit₅, or medium (control). Results represent the percentage ERK1/2 (□) and PKB/AKT (■) phosphorylation (mean ± SEM) compared with medium-treated cells (100%) (3 or more independent experiments). (C) The increase of [Ca²⁺]_i in CHO-CXCR3 cells was measured on stimulation with CXCL10 (♦) or CXCL10-Cit₅ (□). Values represent the mean (± SEM) increase of [Ca²⁺]_i of 3 or more independent experiments with a detection limit at 10 nM (…). (D) Desensitization experiments were performed by rechallenging the CHO-CXCR3 cells with 3 nM of CXCL10 100 seconds after the first stimulus. Results (mean ± SEM) represent the percentage inhibition of the second agonist by the first stimulus in comparison with buffer as first stimulus. Significant differences were calculated using the Mann-Whitney test on paired values (*P < .05, **P < .01 for comparison with buffer; ‡P < .05, ‡‡P < .01 for comparison of CXCL10 with CXCL10-Cit₅) for the corresponding chemokine concentration as depicted in panels A through D.