Pcsk9

Cultured cells were collected with cell lysis buffer (Beyotime, Shanghai, China). Protein samples were separated by precast NuPAGE Novex 4–12 % (w/v) Bis–Tris gels (Life technologies, Carlsbad, CA, USA) and then transferred onto nitrocellulose membrane using the iBlotTM dry blotting system as described by the manufacturer (Invitrogen, Carlsbad, CA, USA). Membranes were blocked in TBST buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1 % tween 20) containing 5 % milk for 1 h at room temperature and incubated with primary antibodies specific for LDLR (Biovision, Mountain View, CA), PCSK9 (Cayman Chemicals, MI), HNF1α (Cell Signaling) and GAPDH (Abcam) overnight at 4 °C, and then incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 2 h at room temperature. Blots were developed using chemoluminescence (ECL, Thermo Fisher Scientific, Waltham, MA, USA) on FluorChem M image system.

Cell lysates were prepared using 1 × RIPA lysis buffer (Biosaesang) supplemented with a protease inhibitor (Roche), cleared by centrifugation at 12,000g, separated by SDS–PAGE, and transferred onto PVDF membranes (Millipore). Primary antibodies were incubated as indicated; overnight at 4 °C in 1 × TBST with 5% skim milk or 5% BSA; PTBP1 (1:1,000, Invitrogen), MAPK14 (1:1,000, Cell Signaling Technology), PCSK9 (1:1,000, Cayman Chemical Company), γ-tubulin (1:1,000, Cell Signaling Technology) and α-tubulin (1:1,000, Cell Signaling Technology).