N6657

Western blot analysis was performed to characterize macrophage polarization, as previously described [34 (link)]. Macrophages were lysed in reducing sample buffer (RIPA with protease inhibitors). The total protein concentrations were determined by means of the BCA Protein Assay Kit (23227, Thermo Scientific). Goat anti-mouse arg-1 (1/1000, sc-271430, Santa Cruz, Heidelberg, Germany) and mouse anti-mouse iNOS (1/500, N6657, Sigma-Aldrich, Bornem, Belgium) was incubated overnight at 4 °C. As a loading control, β-actin (1/2000, 47778, Santa Cruz) was used on the same blot. The PierceTM ECL Plus detection kit (32132, Thermo Scientific) was utilized to assess chemiluminescent signal detection. Images were taken with the AmershamTM Imager 680 (General Electric Company) and used for densitometric analysis with Image Quant TL software.

Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). The protein concentration was determined by the Pierce™ BCA assay kit (ThermoFisher Scientific), according to the manufacturer's guidelines. Samples (4–10 µg) were separated on 7.5% or 12% (dependent on size target) sodium dodecyl sulfate–polyacrylamide gels by electrophoresis (1 h at 200 V) and transferred to a polyvinylidene fluoride membrane (1.5 h at 350 mA, Merck). After blocking using 5% non-fat dry milk in 1× tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T), blots were incubated with primary antibodies overnight at 4 °C. Used antibodies included: mouse anti-Arg1 (1:1000, sc-271430, Santa Cruz Biotechnology), mouse anti-inducible nitric oxide synthase (iNOS, 1:500, N6657, Sigma-Aldrich), and mouse anti-β-actin (1:2000, sc-47778, Santa Cruz). After washing, secondary antibodies (anti-mouse HRP, 1:2000, Dako) were applied for 1 h at RT (room temperature). β-Actin was used as a loading control. Visualization was done using the Pierce™ ECL plus Western blotting substrate (ThermoFisher Scientific). Images were taken with an Amersham Imager 680 (GE Healthcare Bio-Sciences) and analyzed using the ImageQuant TL software. Pictures displayed in the figures were digitally enhanced to improve visibility.