Melanogenesis Protein Detection Protocol

The extraction, separation, and detection of melanogenesis-related proteins, including MITF, tyrosinase, TRP-1, and TRP-2, were performed according to our previous method (Alehaideb et al., 2021 (link)), with modifications. Briefly, after 72 h of incubation, the untreated and treated cells were trypsinized, collected, and rinsed twice with PBS. The cell pellets were extracted once with NP40 buffer, and the protein concentrations were determined using a Qubit^® protein assay kit (Invitrogen). Cell lysates (150.0 μg) were loaded on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were detected and quantified using primary mouse monoclonal antibodies directed against melanogenesis-related proteins including tyrosinase, TRP-1, TRP-2, and MITF. Mouse monoclonal (clone 236–10501, #A11126, Thermo Fisher Scientific) and rabbit polyclonal (#ab18251, Abcam, Cambridge, MA) anti-α tubulin antibodies were loading controls. Infrared fluorescent dye IRDye^® 800CW-conjugated goat anti-mouse antibody (#926–32210) and IRDye^® 680RD-conjugated goat anti-rabbit antibody (#926–68071) were used as secondary antibodies (LI-COR Biosciences, Lincoln, NE). The blots containing the targeted proteins were scanned on the LI-COR Odyssey CLx, and the protein expression levels were analyzed using ImageJ software.