Pparα

Western blotting was performed as previously described (Mukai et al., 2017 (link)). Briefly, C3H10T1/2 cells were lysed with radioimmunoprecipitation assay buffer, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). These membranes were incubated overnight at 4°C with primary antibodies against FABP4 (12802-1-AP; Proteintech, Rosemont, IL, USA), VDR (#12550; Cell Signaling Technology, Danvers, MA, USA), UCP1 (sc-518171; Santa Cruz Biotechnology, Inc., Dallas, TX. USA), CIDEA (13170-1-AP; Proteintech), PPARγ (#2435; Cell Signaling Technology), PPARα (15540-1-AP; Proteintech), FGF21 (ab171941; Abcam, Cambridge, UK) and α/β-tubulin (#2148, Cell Signaling Technology). The membranes were then washed and incubated with horseradish peroxidase–labeled secondary antibodies for 1 h at room temperature. Immunoreactivity was determined using Immobilon-P (Millipore) or Chemi-Lumi One L (Nacalai Tesque), and chemiluminescence was visualized using Amasham Imager 680 (GE Healthcare, Chicago, IL, USA).

Protein expression in HRCPs and mouse retinal tissues was examined by performing western blot (WB) assay. Total protein was extracted from HRCPs and mouse retinal tissues using Total Protein Extraction Kit (Solarbio) as the introduction described. BCA Protein Assay Kit (Solarbio) was used to examine the concentration of proteins. Protein samples were separated by 10% SDS-PAGE electrophoresis. Subsequently, the separated proteins were transferred onto polyvinylidene fluoride membranes (Merck Millipore). After blocked with 5% skim milk, the membranes were incubated with the primary antibodies, PPARα, DNMT1, DNMT3A or DNMT3B (1:1000; Proteintech, Wuhan, China), at 4 °C for 12 h. Next, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:5000; Proteintech). β-actin antibody (1:5000; Proteintech) was used as a reference protein for normalization. The data were analyzed by Image J software.

The protein of liver tissues and HepG2 cells was lysed and extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beijing Beyotime, China), and the concentration was quantified using a BCA assay kit (Solarbio, PC0020, Beijing, China). The denatured protein samples (50 µg) from each group were loaded on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA, USA). Each PVDF membrane was blocked with 1×TBST containing 5% skimmed milk and incubated with primary antibodies at 4°C refrigerator overnight and incubated with secondary antibodies for 1 h in the dark. All the primary antibodies in the Western blot experiment are as follows: PGC1α (1:1000, Proteintech), PPARα (1:1000, Proteintech), PINK1 (1:1000, Proteintech), Parkin (1:1000, Affinity), ATG7 (1:1000, Affinity), SQSTM1/p62 (1:1000, Affinity), FAS (1:1000, ab128856), SREBP-1c (1:1000, A15586), LC3 I/II (1:1000, AB Clonal), and β-actin (1:10000, AB Clonal). The chemiluminescence intensity of all membranes was visualized with enhanced chemiluminescence system. The gray value of protein strips was analyzed using the Image J software.