Ecl chemiluminescence substrate

After the extraction of cells [42 (link)], the extracted protein was quantified by Bradford protein assay. Proteins (30 μg) were separated by 8–15% SDS-PAGE gels via electrophoresis and transferred to poly-vinylidene difluoride (PVDF) membranes for immunoblotting. The membrane was exposed with primary antibodies in 5% skim milk in Tween 20/Tris-buffered saline (T/TBS) overnight at 4 °C. The primary antibody of the membrane was removed by T/TBS solution three times. Then, the immunoblotted membrane was exposed with horseradish peroxidase-conjugated secondary antibody for 2 h at 25 °C. Specific bands were visualized and developed by enhanced chemiluminescence using an ECL chemiluminescence substrate (Santa Cruz Biotechnology, CA, USA).

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).

AKF-D52-treated cells were pelleted by centrifugation (2500 rpm, 10 min, 4 °C). The cell pellet was lysed using protein lysis buffer (Intron, Seoul, Republic of Korea). Total protein concentrations were measured by the Bradford assay. After heat denaturation for 5 min, proteins were resolved by 8–15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% skim milk for 1 h at room temperature and then incubated with specific primary antibodies. After three times washing with Tween 20/Tris-buffered saline (T/TBS), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Following three washes in T/TBS, Specific bands were visualized through Image Quant LAS-4000 (Fujifilm Life Science, Tokyo, Japan) using an ECL chemiluminescence substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All original western blot figures are included in Figures S2–S14.