Macrophage Protein Profiling by Western Blot

Cultured cells (THP-1-derived macrophages and RAW264.7) were lysed in NP-40 lysis buffer. This is composed of 1% NP-40 (Sigma), 20% Glycerol (Santa Cruz^®), 0.2 mM EDTA (Sigma), 40 mM HEPES (Gibco) pH 7.9, 0.5 M NaF (Sigma), 10 mM NaPpi (Sigma), 5 M NaCl (Fisher) and was supplemented with protease inhibitor cocktail (aprotinin, leupeptin, and pepstatin in a 1:1:1 ratio; Sigma), 0.5 M dithiothreitol (Sigma), 10 mg/mL phenylmethanesulfonyl fluoride (Sigma) and 100 mM Na₃VO₄ (Sigma). Debris was removed by centrifugation (Sorvall) at 1500 rpm for 15 min, and proteins were fractionated by SDS-PAGE, 8% polyacrylamide gel (BIO-RAD), and probed for RAGE, JNK, p-Akt, p-STAT3, STAT5b, and GAPDH (Santa Cruz^®). These were visualized with horseradish-peroxidase-coupled 2° antibodies—mouse-IgGκ, goat anti-rabbit IgG, or donkey anti-goat IgG (Santa Cruz^®) [62 (link),64 (link),65 (link)]—developed using western blotting substrates (Thermo Scientific^TM Pierce^TM ECL Plus Western Blotting Substrate, Rockford, IL, USA), and image-captured (FluorChem^TM 8900, ProteinSimple, San Jose, CA, USA). All experiments were done in triplicates. Densitometric analyses (ImageJ, version 1.53a [66 ]) were performed. Each band was quantified five times, and we reported the fold change of each exposure condition compared to media alone (MA), as described [63 (link)].