Anti mouse or anti rabbit horseradish peroxidase conjugated antibodies

Equal amounts of extracted proteins were resolved and transferred to nitrocellulose membranes. These membranes were incubated with antibodies to GDA, tyrosinase, SCF, xanthine oxidase (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA), MITF, bFGF, p-CREB, CREB (rabbit polyclonal; cell signaling technology, Beverly, MA, USA), and β-actin (mouse monoclonal; Sigma-Aldrich). After incubating with appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Thermo Fisher Scientific, Waltham, MA, USA) or with anti-goat horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology), enhanced chemiluminescence solution (Thermo Fisher Scientific) was applied and signals were captured with an image reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). Protein bands were then analyzed by densitometry. Concentrations of bFGF (R&D Systems, Minneapolis, MN, USA) and SCF (Abcam) in culture supernatants were measured using ELISA kits according to the manufacturer’s instructions.

Equal amounts of extracted proteins were resolved and transferred to nitrocellulose membranes. The membranes were incubated with antibodies to IL-1α, IL-1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IL-1ra, cleaved caspase-3 (Cell Signaling Technology, Beverly, MA). After incubating with appropriate anti-mouse or anti-rabbit horseradish peroxidase–conjugated antibodies (Thermo Fisher Scientific, Rockford, IL, USA) and enhanced chemiluminescence solution (Thermo Fisher Scientific), the signals were captured on an Image Reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). To monitor the amount of protein loaded in each lane, the membranes were reprobed with a mouse monoclonal anti-β-actin antibody (Sigma) and were processed as described above. The protein bands were then analyzed via densitometry.