Immunoblot Analysis of Protein Expression

Cells were lysed on ice in a lysis buffer composed of 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) and 5 mM EDTA with protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were centrifuged at 500g for 30 min at 4°C to remove cellular debris. Proteins (25 μg/lane) were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). For the detection of α-syn, membranes were fixed in 4% paraformaldehyde solution as previously described.^{38 (link)} Membranes were immunoblotted for AXL (AF154 R&D Systems at 1:500), MerTK (ab52968, Abcam at 1:500), phosphorylated MerTK (p186-749, Phosphosolutions at 1:1000), α-syn (ab138501, Abcam 1:5000), S129-phosphorylated α-syn (ab51253, Abcam 1:1000), CSF1R (MAB3291, R&D systems; 1:250) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; G8795, Sigma Aldrich at 1:5000) overnight at 4°C, and then with horse radish peroxidase-linked secondary antibodies (1:10 000; Jackson Laboratory) for 1 h. Bands were detected by enhanced chemiluminescence with Clarity Max ECL substrates (Bio-Rad Laboratories) using a ChemiDoc Imaging System (Bio-Rad Laboratories). Image analysis was performed using ImageLab 6.0.1 software (Bio-Rad Laboratories).