Inos sc 650

Mouse macrophage and THP-I whole-cell extracts were isolated for western blot analysis as described previously [37 (link)]. Proteins were resolved by SDS polyacrylamide gel electrophoresis (4–20%) and electrophoretically transferred to polyvinylidene fluoride membranes (0.2 μm pore size). Immunodetection was performed using chemiluminescent detection with the Renaissance Western Blot Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences Inc., Boston, MA). Blots were stripped using the Restore Western Blot Stripping Buffer (Pierce Biotechnology Inc., Rockford, IL) as described by the manufacturer. Purified rabbit polyclonal antibodies to phospho-NFκB p65 (Ser276, Cell Signaling), NFκB p65 (sc-109, Santa Cruz Biotechnology), iNOS (sc-650, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used. Optical densities of antibody-specific bands were determined using Quantity One acquisition and analysis software (Bio-Rad, Hercules, CA).

The RAW 264.7 macrophage cells were seeded in a culture dish at a density of 5 × 10⁵/mL. Cells were pretreated with 50 μM of TCMB (11) for 1 h and then stimulated with LPS 100 ng/mL for 6, 12, and 24 h. After incubation, the cultured media were removed and cells were washed with PBS. Whole protein was extracted by using PRO-PREP protein extraction solution (Intron Biotechnology, Seoul, Republic of Korea). Extracted proteins were quantified by Bio-Rad protein assay using a reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA). The whole protein (25 μg) was separated by SDS-PAGE using 10% acrylamide gel and then transferred to the PVDF membrane. The blots were incubated with iNOS (sc-650, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and COX-2 (No: 160106, Cayman Chemical, Ann Arbor, MI, USA) antibodies diluted in 5% skim milk with 1:1000 and 1:5000 ratio, respectively. After incubation at 4 °C for 18 h, the blots were washed 3 times using TBS/T. The washed blots were then incubated for 2 h with horseradish peroxidase-conjugated secondary antibody diluted in 5% skim milk with 1:2000 ratio and washed 3 times using TBS/T. The washed blots were developed using an ECL chemiluminescence substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The developed blots were then visualized with Amersham Hyperfilm ECL (GE Healthcare Life Sciences, Chicago, IL, USA).