Horseradish peroxidase conjugated anti rabbit

For western blotting, Melan-a cells were lysed with a RIPA buffer and 1X P.I.C (protease inhibitor cocktail) (Sigma-Aldrich). The cell lysates were centrifuged at 13,000×g for 20 min at 4°C. Protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Melan-a cells were lysed with lysis buffer composed of RIPA solution (Noble Bio, Seoul, Korea), 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich), and protease inhibitor cocktail (Sigma-Aldrich) for 1 h at 4°C. The lysates were conducted to centrifugation at 13,000 rpm for 20 min, and the supernatant was used in the analyses. Blots were incubated overnight with Mlph antibody (1:500, Protein Tech Group, Inc, Chicago, IL, USA), or β-actin antibody (1:5,000, Sigma-Aldrich) at 4°C. Blots were then rinsed three times with 1X TBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit (Bethyl Laboratories, Montgomery, AL, USA) or anti-mouse (Bio-Rad, Hercules, CA, USA) antiserum at room temperature for 1 h. Bound antibodies were detected using a WEST-ZOL^® Plus Western Blot Detection System (INtRON Biotechnology, Seoul, Korea) and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). The bands on membranes were detected with chemiluminescence and visualized using a Chemi Doc XRS (Bio-Rad) and FluorChem E (ProteinSimple, San Jose, CA, USA).