Enhanced chemiluminescence western blotting detection kit

HaCaT cells and mouse left-ear tissue were washed with ice-cold PBS, and proteins were isolated in 100 μL of Ceti Lysis buffer (TransLab, Daejeon, Korea) containing protease and phosphatase inhibitors. To evaluate the target protein for NF-κB p65, IκBα, ERK1/2, and STAT1 activation, they were resolved in 10% and 12% polyacrylamide gels, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), blocked in 5% skim milk, and probed with the appropriate primary and secondary antibodies. Membranes were visualized using an enhanced chemiluminescence Western blotting detection kit (BioFact™ Co., Ltd., Daejeon, Korea).

Cell lysates were prepared in lysis buffer (120 mM NaCl, 40 mM Tris [pH 8], and 0.1% Nonidet P-40) on ice for 30 min and centrifuged at 10,000 rpm for 15 min. Supernatants were collected and concentrations were determined at 595 nm using a protein assay kit. Equal amounts of total cellular protein were electrophoresed in 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. After membranes had been blocked in 5% skim milk for 1 h, they were incubated with primary antibodies overnight, then with the appropriate horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Biofact). The integrated optical density for the protein band was calculated by Image-J software version 1.42q (NIH, Bethesda, MD, USA), and then the values were normalized to β-actin level.

After treatment, MCF-7 cells were lysed in lysis buffer (120 mM NaCl, 40 mM Tris [pH 8.0], and 0.1% nonidet P-40) on ice for 30 min and centrifuged at 12,000 rpm for 20 min. Supernatants were collected and protein concentrations were measured using a protein assay kit (Pro-Measure, Intron Biotechnology, Seongnam, Korea). Aliquots of the lysates (50 µg protein) were boiled for 5 min and resolved by using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and incubated with the appropriate primary antibodies. The membranes were incubated with the secondary anti-mouse or anti-rabbit antibody. Finally, the protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Biofact). To investigate multiple protein targets under the same treatment conditions, the blot was stripped and re-probed. Equal sample loading was confirmed by measuring β-actin levels.