Lxr α receptor

In brief, cells of each group were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton-X100, and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum (Beijing Dingguo Changsheng Biotechnology). Cells were incubated with monoclonal antibodies overnight with 4°C. Antibody dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, Irvine, CA, USA). After extensive washing three times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, and anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1 h at room temperature. Nuclear stain 4,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was then used for nuclear staining.

Cells were plated on a six-well plate for 24 h with 10% FBS, then grown in induction medium for 6 days to be used for preparation of whole cell extract. After removing the medium, cells were washed three times with PBS. The cells in each well were then added to 150 µl lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and cracked on ice for 30 min. The mixture was centrifuged at 12,000 revolutions per minute for 20 min at 4°C and the supernatants collected. The protein concentration was determined by a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). The protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with specific primary antibodies, TH, 1:500 (Abcam); LXR α receptor, 1:500 (Abcam); LXR β receptor, 1:500 (GeneTex) were included and overnight at 4°C. The membranes were rinsed three times in TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. After washing three times in TBST, protein signals were visualized by ECL (Bio-Rad, Richmond, CA, USA).