Protein Quantification and Immunoblotting

Tissues or cells were homogenized in 1× RIPA lysis buffer (Millipore, 20-188) with proteinase and phosphatase inhibitors (Roche, 04693124001, 04906845001), and the supernatant was collected after centrifugation at 14,000 × g for 10 min. Protein quantification was performed using BCA protein assay (Pierce, 23227). Total protein for all samples were separated on 4–20% Criterion^TM XT Bis-Tris gels (Bio-Rad), transferred to nitrocellulose membrane (Bio-Rad), and stained with Revert^TM 700 Total Protein Stain Kit (LI-COR, 926-11016) to verify protein concentration and loading accuracy. After blocking with Odyssey blocking buffer (LI-COR, 927-70001), the membrane was incubated with a goat anti mouse/human myeloperoxidase (MPO) antibody (R&D, AF3667), a rabbit anti-mouse phospho (p)-p65 nuclear factor (NF)-κB (CST, 3033S), or a rabbit anti-mouse p65 NF-κB (CST, 8242S) overnight at 4°C, followed by incubation with a donkey anti-goat or anti-rabbit 680RD secondary antibody (LI-COR, 925-68074 or 926-68073) for 45 min at room temperature. The signal was measured at the wavelength of 700 nm with the LI-COR imaging system (Odyssey CLx). The signal of total protein was used as the internal loading control for each lane, and data were quantified as the ratio of MPO to total protein signal (12 (link)). The ratio of p-p65 to p65 was quantified to represent NF-κB activation.