1d imaging software

Activation of p38, ERK1/2, and JNK in human primary keratinocytes or epidermal equivalents was evaluated by Western blot using phospho-specific antibodies, and was normalized to the total level of the relevant proteins using anti-p38, anti-ERK1/2, and anti-JNK1/2 antibodies. Antibodies for phospho-p38 (Thr180/Tyr182), p38, phospho-p44/42 or ERK1/2 (Thr202/Tyr204), p44/42, phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, as well as goat anti-rabbit IgG HRP (horseradish peroxidase) and horse anti-mouse IgG HRP secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). All other reagents for Western blot were purchased from Sigma (St. Louis, MO, USA), and blotting equipment was purchased from Bio-Rad (Hercules, CA, USA). The separated proteins were visualized by an ECL kit according to the manufacturer’s protocol (GE Healthcare Bio-Sciences Co., Piscataway, NJ, USA) and quantitated by densitometric analysis using a Kodak Gel Logic 100 Imaging System and Kodak 1D Imaging Software (Eastman Kodak Co., New Haven, CT, USA).