Ab140606

The following antibodies were used in this study. TFEB (A303-673A-T, Bethyl Laboratories), MITF (ab140606, abcam), LC3 (PM036, MBL International), SQSTM1/P62 (ab109012, abcam).

After treatment with various compounds (0.2% DMSO served as a control), the cells were lysed with cell lysis buffer. The antibodies used were antibodies against Akt (9272S; CST, Danvers, USA), pAktSer473 (4060S; CST), Met (ab216330; Abcam, Cambridge, UK), β-catenin (8480S; CST), non-phospho (active) β-catenin (Ser45) (19807S; CST), non-phospho (active) β-catenin (Ser33/37/Thr41) (8814S; CST), MiTF (ab140606; Abcam), TRP2/DCT (ab221144; Abcam), and β-actin (9272S; CST), and the target bands were detected with a chemiluminescence system (Clinx Science Instruments, Shanghai, China).

B16 cells (5 × 10⁴ cells/Petri dishes) were seeded and incubated at 37 °C in an incubator with 5% CO₂. After 24 h incubation, the growth medium was replaced with a fresh growth medium with or without 10 μg argan leaf saponin and 100 μM arbutin, and incubated further for 24 h and 48 h. After the specified incubation time, the protein samples were extracted using a RIPA (radio immunoprecipitation assay) lysis buffer (SIGMA; St. Louis, MO, USA) with 0.1% protease inhibitor cocktail (SIGMA; St. Louis, MO, USA) following the manufacturer’s instructions. The protein samples were then loaded into 10% SDS-polyacrylamide gel and subjected to electrophoresis (SDS-PAGE). The proteins in the gel were transferred onto a PVDF membrane and incubated in specific primary antibodies against MITF (ab140606, Abcam, Waltham, MA, USA), TYR (ab180753, Abcam, Waltham, MA, USA), TYRP1 (ab221144, Abcam, Waltham, MA, USA), DCT (ab178676, Abcam, Waltham, MA, USA), and GAPDH (ab181602, ab9485, Abcam, Waltham, MA, USA). Membranes were washed with PBS with Tween-20 (PBST) before incubation with goat anti-mouse IRDye 680LT or goat anti-rabbit IRDye 800CW (LI-COR) secondary antibodies at room temperature.