Enhanced chemiluminescent substrate

After 24 hours of treatment, cells grown in either 100 or 150 mm plates were washed three times with PBS and harvested into lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% NP-40, pH=7.4 plus protease and phosphatase inhibitors) on ice. The lysates were then centrifuged at 23,000 x g for 25 minutes at 4°C, and each supernatant was transferred to another microcentrifuge tube. A BCA kit (Pierce) was used to determine protein concentration, and samples were then equalized using lysis buffer. For SDS-PAGE, samples containing 30 μg of protein were separated on 10-well 10% Mini-PROTEAN® TGX™ pre-cast gels (Bio-Rad) and then transferred to polyvinyl difluoride membranes using the Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The membranes were blocked for 1 hour in 5% bovine serum albumin (BSA) and then incubated with primary antibody for 1-2 hours according to the manufacturer's recommended dilution (typically 1:1000). After washing, the membrane was incubated with a horseradish peroxidase conjugated secondary antibody for 1 hour (1:30,000). After washing, 1 mL of an enhanced chemiluminescent substrate (Thermo Scientific) was applied to the membrane and imaging was carried out in a dark room with HyBlot® CL autoradiography film (Denville Scientific).

Human ORS and DP cells or skin tissue were lysed (RIPA lysis buffer; #20‐188; Merck Millipore). The obtained protein was then separated by 8% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Amersham). The protein transferred membranes were incubated overnight with the specific antibodies. The following antibodies were used: β‐actin (#MA5‐15739, 1:5,000; Thermo Fisher), phospho‐ERK1/2 (#9101, 1:1,000; Cell Signaling), total ERK1/2 (#9102, 1:1,000; Cell Signaling), phospho‐AMPK (#2535, 1:1,000; Cell Signaling), and total AMPK (#2532, 1:1,000; Cell Signaling). Then, the membranes were washed and incubated with anti‐rabbit IgG or anti‐mouse IgG antibodies (horseradish peroxidase‐conjugated, GTX213110, GTX213111, 1:10,000; GeneTex) at 25°C for 1 h. Antibody‐antigen complexes detected by enhanced chemiluminescent substrate (Thermo Fisher) were captured and quantified by Amersham imager 680 systems (GE Healthcare).

Total proteins were extracted from A549 and PC9 cells by using RIPA buffer (Cell Signaling Technologies, USA) on ice for 30 min. However, For detection of cytochrome c release from mitochondria, the mitochondria fraction and cytoplasm fraction was separated by using Mitochondria/Cytosol Fraction Kit (BioVision, USA). After protein extraction, equal amount of proteins were separated by SDS-PAGE, and then transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% skimmed milk and probed with corresponding primary antibodies. Next, proteins on the membranes were probed with HRP-conjugated secondary antibodies, and then visualized with enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc, USA).