Inhibiting CSF1R signaling in macrophages with the pharmacologic inhibitor GW2580. (A) Immunoprecipitation and Western blot analysis of Raw264.7 murine macrophage cells stimulated with 10 ng/mL CSF-1 for 20 minutes in the absence or presence of 10, 100, or 1000nM GW2580. Blot was probed for tyrosine phosphorylation (pTyr), stripped, and reprobed for total CSF1R. (B) BMDMs were stimulated with CSF-1 in the absence (□) or presence of 10nM (▵), 100nM (◇), or 1000nM (○) GW2580. At the indicated time points, cell viability was measured using the CCK-8 assay and compared with unstimulated control. (C) BMDMs were seeded on 8-μm transwell inserts, and CSF-1 was added to the lower chamber in the absence or presence of 1000nM GW2580 (added to both chambers). Cells were allowed to migrate toward CSF-1 for 6 hours and then fixed and stained with DAPI. Representative images are shown and migrated cells were quantified using ImageJ software (□, control, no CSF-1; , CSF-1; ■, CSF-1/GW2580). Scale bar represents 100 μm.