G box image station ichemi xl device

Control (20 nM) or miR-4669 mimic (10 or 20 nM)-transfected Hep3B cells were cultured for 72h, and GAPDH levels in the culture medium were semi-quantified using western blot analysis. Briefly, proteins in culture media (10 μL) were fractionated and transferred onto a PVDF membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The membrane was immunoprobed with a rabbit anti-GAPDH monoclonal antibody (×500 dilution, Abcam, Cambridge, UK) followed by incubation with HRP-conjugated anti-rabbit IgG (×5000 dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Signals were visualized using an Immunobilon Forte Western HRP substrate (MilliporeSigma), and signal intensity was quantified using a G:BOX Image Station iChemi XL device (SYNGENE, Cambridge, UK).

The proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad). The membranes were blocked with 5% (w/v) skim milk/1% (v/v) Tween 20 in PBS for 30 min at room temperature and incubated overnight with the appropriate primary antibody: Insig 2 from Biorbyt (Biorbyt Llc, SF, CA, USA, 1:500 dilution); SCAP from Bioss (Bioss Inc., Woburn, MA, USA, 1:1000 dilution); SREBP1 antibody from BioVision (BioVision Inc., Milpitas, CA, USA, 1:500 dilution); NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA,USA, 1:1000 dilution); phospho-NF-κB p65 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:1000 dilution); anti-β-actin antibody from Millipore (Millipore Corporation, Bedford, MA, USA, 1:10000 dilution) at 4 °C. Detection was performed using an enhanced chemiluminescence (ECL) detection kit (Millipore). Images were captured using a G:BOX Image Station iChemi XL device (SYNGENE, Cambridge, UK), and the relevant bands were quantified by densitometry using GeneTools (SYNGENE).