Effect of cinnamon water extract on monocyte-to-macrophage differentiation and scavenger receptor activity

Kang, Hee; Park, Sung-Hyun; Yun, Jeong-Moon; Nam, Tae-Gyu; Kim, Young-Eun; Kim, Dae-Ok; Kim, Youn Jung

THP-1 cells (1 × 10 7 cells) were pretreated with CWE or U0126 (20 μM) for 1 h prior to exposure to PMA for 30 min for ERK1/2 activity or for 72 h for SRA synthesis. Mouse macrophages (5 × 10 6 cells) were cultured with M-CSF (25 ng/ml) (R&D Systems, MN, USA) in the presence of CWE for 24 h. Cells were rinsed in cold PBS and then lysed on ice in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 20 mM NaF; 0.5% NP-40; and 1% Triton X-100) containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Roche Diagnostics, Germany). After centrifugation at 13,000 × g for 10 min, supernatants were collect, and protein concentrations were determined using the Bradford protein assay reagent (Bio-Rad, USA). The samples were separated on an 8% or 10% SDS-polyacrylamide gel and were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. The membranes were incubated with antibodies against human SRA, actin, GAPDH (Santa Cruz Biotechnology, CA, USA), mouse SRA (R&D Systems), extracellular signalrelated kinase (ERK) 1/2, or phospho-ERK1/2 ( Cell Signaling Technology, MA, USA ) diluted in 5% skim milk in TBST overnight at 4°C. The blots were washed with TBST and incubated for 1 h with anti-goat, mouse or rabbit horseradish peroxidase-conjugated antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence using BioFx (SurModics, MN, USA).