Western-blot analysis for cathepsin D and Nrf-2

Form the homogenized skin, 50 µg protein lysates from each sample were mixed with 2X loading buffer (130 mM Tris–HCl, pH 8.0, 30% (v/v) Glycerol, 4.6% (w/v) SDS, 0.02% Bromophenol blue, 2% DTT), boiled for 5 min then cooled at 4 ℃. Samples were separated on 12% SDS-PAGE mini-gel (1.6 ml H2O, 1.5 M Tris pH 8.8, 1.3 ml, 30% Acrylamide acrylamide/bisacrylamide (29:1 mix in 100 ml) 2.0 ml, 10% SDS, 10% APS, TEMED 2 ml) and run at 120 V. Proteins were transferred to a nitrocellulose membrane at 22 V overnight at 4 ℃. The membrane was washed 3 times with Tris-buffered saline containing Tween 20 (TBST) (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and then incubated in blocking buffer (TBST containing 5% non-fat dry milk) for 1 h at RT and then incubated overnight with primary antibodies for Cathepsin D, Nrf-2, or β-actin diluted with TBST and 5% bovine serum albumin (BSA). After 3 times wash with TBST, the membrane was incubated for 1 h at RT with secondary antibody. Membranes were again washed three times with TBST, and bands were detected by alkaline phosphatase solution, then bands were quantified using Image J quantification software (Burnette 1981).