Fusion fx device

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).

Protein was extracted from whole-cell lysates using radioimmunoprecipitation assay buffer (30 mM Tris, 0.5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS) supplemented with 10% protease and phosphatase inhibitors (Roche, Switzerland). To ascertain protein concentration, a bicinchoninic acid kit (Thermo Fischer, USA) was used. Western blots were performed as described previously (El-Armouche et al. 2008 (link)). A 20 μg of a whole-cell protein was separated on a 10% polyacrylamide gel and then transferred to a nitrocellulose membrane. Immunodetection was performed with a Fusion FX device (Vilber Lourmat Deutschland GmbH, Germany). Table 1 provides a list of antibodies and respective concentrations used in this study.

Cell lysis, SDS-PAGE, and immunoblotting were performed using standard procedures. A measure of 40 μg of proteins were loaded per lane for Western blot and samples underwent electrophoresis through an 8% polyacrylamide gel at 125 mV for 1 h 30. Primary antibodies used for Western blotting are listed in Table S2. Secondary antibodies were HRP-conjugated goat anti-mouse (GE healthcare, Chicago, Il, USA) and goat anti-rabbit (Dako-Agilent, Santa Clara, CA, USA) antibodies. Protein signals were revealed by SuperSignal West Pico and Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and captured with the Fusion FX device (Vilber Lourmat, Marne-la-Vallée, France).