Nbp1 00876

Frozen rat liver was homogenized in 20 mM Tris–HCl buffer (pH 7.8) containing 0.2% Triton X-100 and protease inhibitor cocktail (Sigma, USA), and then centrifuged (15 000 × g, 20 min, 20 °C). Aliquots of the obtained supernatants containing 10 µg of protein were separated by 10% SDS-PAGE and electroblotted to Immuno-Blot™ PVDF Membrane (Bio-Rad Laboratories, Hercules CA, USA). The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich). Secondary HRP-conjugated antibodies were obtained from Sigma Aldrich (A0545, A9044, A5420). The reactions were visualized with a SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA). The bands (visible on the film after the chemiluminescent detection) were compared to molecular mass protein markers (SM1811) obtained from Fermentas, visible on the membrane after electroblotting. The film was adjusted to the membrane in such way that the membrane edges were visible on the film. Blots were analyzed using Quantity One program, version 4,0 (Bio-Rad).