For the zymosan-induced peritonitis experiments, peritoneal exudate cells harvested at the times indicated in the figure legend were washed with fluorescence-activated cell sorting (FACS) staining buffer [PBS containing 3% (v/v) FBS]. The cells were incubated for 5 min at 4°C with Fc Block (BD Biosciences) and then labeled with Pacific Blue–Ly6G (clone 1A8, eBioscience) to detect polymorphonuclear neutrophils. To detect p-CaMKII in exudate macrophages, cells were stained with PE-F4/80 (clone BM8, eBioscience) for 30 min at 4°C to stain macrophages, which was followed by fixation and permeabilization of the cells. Permeabilized cells were then incubated with rabbit anti–p-CaMKII for 1 hour at 4°C and then with Alexa Fluor 647–conjugated goat anti-rabbit secondary antibody for 30 min at 4°C. For the flow cytometric assay of p-ERK and p-CaMKII in WT MerTK–expressing versus Y872F MerTK–expressing Mertk−/− macrophages, the cells were first incubated with allophycocyanin (APC)–MerTK (clone #125518, R&D Systems) to label transduced macrophages. After fixation and permeabilization, the cells were incubated with rabbit anti–p-ERK or anti–p-CaMKII antibodies, which was followed by incubation with PE-conjugated anti-rabbit secondary antibody. The cells were suspended in FACS buffer and analyzed for the MFI of p-ERK and p-CaMKII in APC-MerTK+ cells gated using a FACSCanto II (BD Biosciences) flow cytometer and FlowJo software.

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