In vitro Assessment of CSF1R Phosphorylation

The N13 murine microglia cell line (21) was cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum and 50 U/mL penicillin/ streptomycin (Thermo Fisher Scientific). Cells were maintained in T75 flasks at 37°C in a 5% CO₂ humidified atmosphere. Cells were plated at a density of 2 × 10⁵ cells/cm² in 6-well-plates and cultured overnight to allow adherence. Cells were plated at a density of 2 × 10⁵ cells/cm² in 6-well-plates and cultured overnight to allow adherence. Cells were kept in serum-free medium for 4 h prior to stimulation and then incubated for the indicated time points (5 or 10 min) with recombinant CSF-1 (50 or 100 ng/mL), IL-34 (50 or 100 ng/mL) (R&D Systems) or LPS (1 μg/mL) as a negative control for CSF1R pathway activation (22, 23), after which cells were immediately lysed in RIPA buffer (Thermo Fisher Scientific), supplemented with protease and phosphatase inhibitor cocktails (Roche, Thermo Fisher Scientific). Protein lysates were concentrated using Microcon-10kDa Centrifugal Filter Units (Merck Millipore), according to manufacturer's instructions and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were subjected to SDS-PAGE and Western blot.