Dnmt3b

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.

Cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The total protein concentrations of the lysates were determined using the Bio-Rad protein assay kit. Equal amounts of protein were denatured with loading buffer (Beyotime) at 100°C for 10 min, then loaded in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis for electrophoresis and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, United States). The membrane was blocked in tris-buffered saline containing 5% bovine serum albumin (MP Biomedical, United States) for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C. The primary antibodies included AKT2, CD133, DNMT3B, MYC, PIK3R1, RBL1 (1:1,000, Proteintech, United States), Bcl-2 (1:1,000, Affinity Biosciences, United States), and GAPDH (1:2,000, ZSGB-BIO, China). After washing with tris-buffered saline twice, the membrane was incubated with the appropriate horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. The secondary antibody conjugated with horseradish peroxidase is goat anti-rabbit immunoglobulin G (1:5,000, ZSGB-BIO). Immunoblots were visualized using enhanced chemiluminescence detection system according to the manufacturer’s protocol.

The 30-µg protein extracts were mixed with a sample buffer, boiled for 10 min, and followed by electrophoresis using 8–15% sodium dodecyl sulfate-polyacrylamide gels. We transferred the proteins in the gels to a polyvinylidene difluoride membrane and incubated the blots with primary antibodies against, α-SMA (abcam, JHY, Cambridge, UK), DNMT1 (Santa Cruz, CA, USA), DNMT3a (Santa Cruz, CA, USA), DNMT3b (Santa Cruz, CA, USA), p-PI3K (PROTEINTECH, Rosemont, IL, USA), p-SMAD3 (abcam, JHY, Cambridge, UK), LC3B II (Cell signaling, Danvers, MA, USA), and GAPDH (PROTEINTECH, IL, USA) for protein control. After washing the blots with tris-buffered saline and incubating them with horseradish peroxidase-coupled anti-rabbit immunoglobulin-G antibodies (dilution, 1:5000), HRP anti-mouse immunoglobulin-G antibodies (dilution, 1:10,000), and HRP anti-goat immunoglobulin-G antibodies (dilution, 1:10,000) at room temperature for 1 h, we developed them with enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Uppsala, Sweden), exposed them to film, and quantified the signals using densitometry.