Protein Lysate Analysis in Stimulated BMDMs

Total protein lysates from stimulated BMDMs were collected using sodium dodecyl sulfate (SDS) lysis buffer (2% (w/v) in 125 mM Tris). Protein concentrations were measured using Pierce BCA protein assay kit (Thermo Fisher Scientific, Merelbeke, Belgium) according to the manufacturer’s instructions and iMARK Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). Protein samples (10 µg) were separated on 12% or 7.5% (for iNOS) SDS gels for 45 min at 200 V. Western blot was performed as previously described [27 (link),52 (link)]. The primary antibodies used were as follows: rabbit anti-mouse acetylated histone 3 lysine 9 (1/2000; Cell signalling, Leiden, The Netherlands), rabbit anti-mouse acetylated histone 3 lysine 27 (1/1000; Cell signalling), goat anti-mouse arginase-1 (Arg-1; 1/1000; Santa Cruz Technologies, Dallas, TX, USA), mouse anti-mouse inducible nitric oxide synthase (iNOS; 1/500; Sigma-Aldrich), and mouse anti-mouse -actin (1/5000, Santa Cruz). The measured values were normalized to the level of beta-actin or total histone 3, as internal controls. To improve readability of the images, the contrast was enhanced (same % as beta-actin per blot) and the images were cut in the representative protein blots in Figure 1 and Figure 2.