To detect IκBα by Western blot, 20-μg protein of B16F10 cell lysate was loaded onto to a 4 to 20% precast gel (Bio-Rad no. 456-1094, Bio-Rad Laboratories, Hercules, CA) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with blocking buffer [5% BSA in tris-buffered saline Tween-20 (TBST) buffer] for 2 hours at room temperature. The membrane was then incubated with an IκBα antibody (Cell Signaling no. 9242, Cell Signaling Technology, Danvers, MA) diluted at 1:1000 in blocking buffer overnight at 4°C. The membrane was washed with TBST buffer three times and incubated with anti-rabbit antibody diluted at 1:5000 (Bio-Rad no. 72-1019, Bio-Rad, Hercules, CA) at room temperature for 1 hour. After three washes, the membrane was incubated with enhanced chemiluminescence substrate (ECL; Bio-Rad no. 1705061, Bio-Rad, Hercules, CA) for 5 min and imaged using the Azure c600 Western blot imaging system (Azure Biosystems, Dublin, CA). The membrane was stripped and subsequently incubated with β-actin antibody at a dilution of 1:100,000. To detect MPO by Western blot, 30-μg protein of B16F10 cell lysate was loaded on a 4 to 20% precast gel, transferred to a PVDF membrane, and blocked in 5% nonfat milk at room temperature for 2 hours. The membrane was incubated overnight with MPO antibody diluted at 1:3000 (AF3667, R&D Systems Inc., Minneapolis, MN, USA). The membrane was washed with TBST buffer three times and incubated with anti-goat antibody diluted at 1:5000 (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 hour. After three washes, the membrane was incubated with ECL for 5 min and imaged using the Azure c600 Western blot imaging system. The membrane was stripped and subsequently incubated with GAPDH (glyceraldehyde phosphate dehydrogenase) antibody (R&D Systems Inc., Minneapolis, MN, USA) at a dilution of 1:10,000.

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