Western Blotting

Primary microglia were lysed in a buffer containing 50 mM Tris–HCl, pH 7.2, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 40 mM NaF, 2.175 mM NaVO₄, 0.1% SDS, 0.1% aprotinin and 1 mM PMSF. Ten micrograms of protein underwent SDS–PAGE following transfer on nitrocellulose membranes. Bands were detected using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA) on a Chemidoc digital imaging machine. We utilized an anti-iNOS rabbit primary antibody (1:200 in 1% BSA; Cayman Chemical, Ann Arbor, MI, USA) followed by a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:3,000; Bio-Rad, Hercules, CA, USA). Internal loading control was performed by anti-α-Tubulin mouse primary antibody (1:500 in 1% BSA; Sigma-Adrich, St. Louis, MO, USA) followed by a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:1,000; Sigma-Aldrich, St. Louis, MO, USA; see Supplementary Figure S2 for antibody specificity). Densitometry quantification was performed with ImageJ Software (NIH) and expressed as ratio of iNOS to α-tubulin.