Rabbit anti β actin antibody

Dulbecco's modified Eagle medium (DMEM) and 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) were purchased from Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan). The cell proliferation reagent water-soluble tetrazolium salt (WST-1) was purchased from Roche (Mannheim, Germany). Bovine serum albumin (BSA) and TRI reagent were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Mouse antihuman tyrosinase (T311) antibody, mouse antihuman TRP1 (G-9) antibody, mouse antihuman TRP2/DCT (C-9) antibody, and mouse antihuman microphthalmia-associated transcription factor (MITF; D-9) antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-β-actin antibody was purchased from BioLegend (San Diego, CA, USA). Donkey antimouse IgG-HRP antibody, donkey antirabbit IgG-HRP antibody, and enhanced chemiluminescence (ECL) prime Western blotting detection reagents were purchased from GE Healthcare (Waukesha, WI, USA). A high-capacity cDNA synthesis kit was purchased from Applied Biosystems (Foster City, CA, USA). SsoAdvanced Universal SYBR Green Supermix was purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Human epidermal keratinocytes were lysed in RIPA buffer (10 mM Tris-HCl, 1% NP-40, 0.1% SDS, 150 mM NaCl and 1 mM EDTA) containing protease inhibitor cocktail (Santa Cruz Biotechnology). The homogenates were centrifuged at 10,000 rpm, 4°C for 20 min. The supernatants were mixed with NuPAGE LDS sample buffer (Invitrogen) and heated for 3 min at 100°C. The samples were electrophoretically separated by the NuPAGE System (Invitrogen) using a 4–12% Bis-Tris gel, and electroblotted onto a PVDF membrane using iBlot Dry Blotting System (Invitrogen). The membrane was blocked with Western Breeze Blocking Solution (Invitrogen) for 30 min at RT, and incubated with goat anti-Ob-R antibody (1:250; R&D Systems) or rabbit anti-β-actin antibody (1:1000; Biolegend, California, USA) for 1 h at RT. The membrane was washed several times with Western Breeze Wash Solution (Invitrogen) at RT. Thereafter, the membrane was incubated with Secondary Antibody Solution (Invitrogen) for 30 min at RT. After additional washes, leptin and β-actin proteins were visualized using Western Breeze Chemiluminescent Substrate (Invitrogen) and ECL mini-camera (Amersham Pharmacia Biotech, Poole, UK).