Sc 20035

Antibodies against total LKB1(liver kinase B1) (3047), phospho (Serine 428)-LKB1 (3482), total AMPKα (5831), phospho (threonine 172)-AMPKα (2535), total ACC (3662), phospho (serine 79)-ACC (acetyl-CoA carboxylase) (3661), p44/42 MAPK (4695), Phospho-p44/42 MAPK (4376), p38 MAPK (9212), Phospho-p38 MAPK (9211), JNK (9252), Phospho-SAPK/JNK (4668), CREB (9197) and Phospho-CREB (9198) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against tyrosinase related protein 1 (Trp1, sc-166857), tyrosinase related protein 2 (Trp2, sc-74439), tyrosinase (sc-20035), microphthalmia-associated transcription factor (MITF, sc-52938), β-actin (sc-47778), and c-Myc tag (sc-47694) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody against melanocortin 1 receptor (MC1R, PA5-21911) and all cell culture media and supplements were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Gibco (Grand Island, NY, USA). α-MSH, Arbutin, AICAR, compound C, PD98059, SP600125, SB203580, H89, A-769662, BAPTA-AM and STO-609 were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The protein was extracted from the cells as described for the tyrosinase activity assay. Proteins were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with PBS containing 0.1% Tween 20 (PBST) and 3% BSA for 1 h and then incubated with an anti-MITF antibody (1:1000, ab12039; Abcam), anti-tyrosinase antibody (1:500, sc-20035; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TRP1 antibody (1:1000, ab235447; Abcam) at 4 °C. The next day, membranes were washed 3 times with 0.1% PBST and incubated for 1 h with the respective secondary antibodies. After 4 washes with 0.1% PBST, the protein-of-interest was detected using a Bio-Image Analysis System (Amersham Imager 600; Fuji Photo Film Co., Ltd., Tokyo, Japan).