Chemiimager analyzer system

Cells were washed three times with phosphate buffered saline (PBS), scraped off plates, and lysed with lysis buffer (1% Triton X-100, 1% deoxycholate). The protein concentration of lysates was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA), and equal amounts of protein were separated electrophoretically using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes (Bio-Rad Laboratories). Membranes were blocked in 5% skim milk in PBS and incubated with primary antibodies against HO-1, COX-2, iNOS, and β-actin diluted 1:500 in 1% skim milk in PBS overnight at 4°C. The blots were then incubated with a peroxidase-conjugated goat antirabbit IgG diluted 1:5000 for 1 h. Protein bands were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, San Jose, CA, USA) and analyzed using a chemiImager analyzer system (Alpha Innotech, San Leandro, CA, USA).

After the cells were treated in the same manner as the method for measuring cell viability, total protein from HaCaT cell lysates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using 12% gels. Protein bands were then transferred to PVDF membranes (BioRad, Hercules, CA, USA), which were blocked with 5% skim milk in PBS then incubated with a 1:1000 v/v dilution of primary antibodies against COX-2, PGE₂, PCOL1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Cell Signaling, Danvers, MA, USA), MMP-1 and Elastin (Abcam, Cambridge, MA, USA) in PBS with 1% skim milk overnight at 4°C. The blots were then incubated with peroxidase-conjugated goat anti-rabbit IgG (PGE₂, β-actin, MMP-1, Elastin) and were then incubated with peroxidase-conjugated goat anti-mouseIgG (COX-2, PCOL1) (1:10,000 v/v, Millipore, CA, USA) for 1 hr. The immunoreactions were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, CA, USA) on a ChemiImager analyzer system (Alpha Innotech, San Leandro, CA, USA).