Stimulated BMDMs were lysed in SDS lysis buffer (2% (w/v) in 125 mM Tris) to collect total protein lysates. Using Pierce BCA protein assay kit (Thermo Fisher Scientific) according to manufacturer's instructions and iMARK Microplate Reader (Bio-Rad Laboratories, Hercules, U.S.A.), protein concentrations were measured. Protein samples (10 µg) were separated on 12% or on 7.5% (for iNOS) SDS gels for 45 minutes at 200 V. The western blot was performed as previously described [6 (link)]. The primary antibodies used were: rabbit anti-mouse acetylated histone 3 (1/2000; Cell signalling, Leiden, The Netherlands), goat anti-mouse arginase-1 (Arg-1; 1/1000; Santa Cruz Technologies, Dallas, U.S.A.), mouse anti-mouse inducible nitric oxide synthase (iNOS; 1/500; Sigma-Aldrich), mouse anti-mouse β-actin (1/5000, Santa Cruz). The measured values were normalized to the level of β-actin or total histone 3 and 4, as internal controls. Selected blots (Fig. 1B , 5D and 6B ), which derive each from the same membrane have been cut and reordered to increase readability.
Mouse anti mouse inducible nitric oxide synthase inos
Manufactured by Merck Group
Sourced in United States
The Mouse anti-mouse inducible nitric oxide synthase (iNOS) is a laboratory equipment product. It is used for the detection and quantification of inducible nitric oxide synthase, an enzyme involved in the production of nitric oxide.
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2 protocols using mouse anti mouse inducible nitric oxide synthase inos
1
Western Blot of Stimulated BMDMs
2
Protein Lysate Analysis in Stimulated BMDMs
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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 .
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