Westernbreeze wash solution

For each fraction, 27 μL (6% of total volume) was suspended with 8 μL of 4x Laemmli Sample Buffer (BioRad, 1610747). The samples were boiled at 95 °C for 5 min and loaded to 10% polyacrylamide gel. After migration, proteins were transferred onto a PVDF membrane using a Trans-Blot SD Semi-Dry Transfer Cell (BioRad) at 15 V for 30 min. Then the different membranes were blocked for 1 h at room temperature in WesternBreeze™ Solution (Invitrogen, WB7050) and incubated with the respective primary antibody (1:50 in WesternBreeze; 1:10,000 for anti-Aldolase) at 4 °C overnight with regular shaking. After 3 washes with WesternBreeze™ Wash Solution (Invitrogen, 46-7005), the blots were probed with HRP-labeled Goat anti-Rabbit IgG (H + L) (1:10,000, Novex^TM, A16104). Next, Clarity^TM Western ECL Substrate (Bio-Rad, 1705060) was applied to develop the membranes. For each antibody, all 50 fractions distributed over four membranes (25 fractions per condition) were analyzed simultaneously by a ChemiDoc™ (BioRad). Relative protein abundance was normalized using Image Lab software (Bio-Rad).

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).