Bradford assay

M-CSF differentiated macrophages were starved for 4h in medium then stimulated with hIgG1 or αChemR23 (10µg/ml) for different times. Cells were lysed with 200µl of RIPA (Sigma-Aldrich) buffer 1X supplemented with protease inhibitor cocktail (Fast protease inhibitor, Sigma-Aldrich) and phosphatase inhibitors (Phos STOP, Roche). Cell lysates were centrifuged for 25min at 800g at 4°C to remove debris and the supernatants were stored at -80°C. Proteins were quantified using Bradford assay (Interchim). 7µg of proteins were loaded in 4-20% Mini-PROTEAN^® TGX™ Precast Protein Gel (#4561093, Biorad) and then transferred onto a nitrocellulose membrane. After 2h of saturation in 5% milk/TBS-Tween 0,05%, the membranes were incubated with primary antibodies anti–phospho(Thr202/Tyr204) - ERK1/2 (p44/42 MAPK) (#4370, Cell Signaling) (1:1000), anti- ERK1/2 (p44/42 MAPK) (#9102S, Cell Signaling) or anti–phospho(Ser473) -AKT (#4051S, Cell Signaling) (1:1000), anti-AKT (#4691S, Cell Signaling) or anti-Actin M(AB1501 Millipore) for 1h at room temperature. Proteins were incubated with Goat anti-Rabbit (#111-001-003) or Goat anti-Mouse (115-035-008) secondary antibodies (Jackson ImmunoResearch) for 1h at room temperature and then revealed with the Immobile Western Chemiluminescent HRP Substrate (WBKLS0500, EMD Millipore). The data were analyzed with the Fusion FX device (Vilber).

Cells were harvested at 10⁶/ml per condition, washed in 1X PBS and lysed on ice in RIPA buffer (Sigma) containing protease and phosphatase inhibitors cocktail (Thermofisher). For phosphoblot experiments, cells were starved overnight without EPO, before re‐stimulation with 10 IU/ml EPO for 5 min. For protein localization, nuclear and cytoplasmic extracts were purified according to standard procedure. Lysates were gently sonicated, centrifuged and protein concentration was quantified using Bradford assay (Interchim). After standard procedures of SDS‐polyacrylamide gel electrophoresis, nitrocellulose membrane transfer and 5% non‐fat milk blocking, blotting was performed using 1/1000 diluted primary antibodies (references in Table S2). Bound primary antibodies were detected using anti‐mouse (Sigma‐Aldrich) or anti‐rabbit (Thermofisher) horseradish peroxidase‐ conjugated secondary antibody. Revelation was performed by enhanced chemiluminescent substrate, SuperSignal West Femto or Pico Substrates (Thermofisher), and signals were visualized using Chemidoc Device (BioRad). Images were analysed using ImageLab software.