Fig 1: EphA2 Activates the MEK-ERK MAPK Module to Prime p47phox(A) Intracellular ROS accumulation measured by mean fluorescence (FL) intensity in wild-type and EphA2−/− neutrophils after45 min of infection with opsonized C. albicans yeast. Results are median ± interquartile range of neutrophils from 3 mice per strain, each tested in duplicate. ****p < 0.0001 (Mann-Whitney test corrected for multiple comparisons).(B) Effects of EphA2 on localization of p47phox in neutrophils infected with C. albicans. Wild-type and EphA2−/− neutrophils were incubated with opsonized GFP-expressing C. albicans yeast for 30 min, fixed, and stained for p47phox (red) and F-actin (blue). Scale bar, 10 μm.(C) Percentage of phagosomes containing C. albicans and surrounded by p47phox. Results are median ± interquartile of 60 neutrophils per mouse strain in three independent experiments. ****p < 0.01 (Mann-Whitney test).(D) Representative immunoblot of MEK½ and ERK½ phosphorylation in wild-type and EphA2−/− neutrophils that had been infected with yeast-phase C. albicans for 30 min.(E) Representative immunoblot of PKC-δ phosphorylation in wild-type and EphA2−/− neutrophils that had been infected with yeast-phase C. albicans SC5314 for 30 min with a 5:1 ratio.(F) Representative immunoblot of ERK½ phosphorylation in wild-type and EphA2−/− neutrophils stimulated with 50 nM PMA for 30 min.(G) Intracellular ROS accumulation (mean fluorescence [FL] intensity) of wild-type and EphA2−/− neutrophil stimulated with PMA for 45 min. Results are median ± interquartile range of neutrophils from 3 mice per strain, each tested in duplicate. ****p < 0.0001 (Mann-Whitney test corrected for multiple comparisons).(H) Effects of inhibiting ERK with the specific inhibitor SCH7729884 on the killing of C. albicans by bone marrow neutrophils isolated from wild-type mice. The percentage of organisms killed was determined by colony counting. Results are median ± interquartile from 4 experiments in triplicate. **p < 0.01, ****p < 0.0001 (Mann-Whitney test).(I) Representative immunoblot analysis of Ser345 p47phox phosphorylation in wild-type and EphA2−/− neutrophils that had been infected with yeast-phase C. albicans for 30 min.(J) β-glucan induces p47phox priming. Wild-type BM neutrophils were incubated with 10 μg/mL of Sepharose IgG beads and/or 10 μg/mL of zymosan (depleted) for 30 min. Representative immunoblot analysis of Ser345 p47phox phosphorylation.See also Figure S5.