Tanon 5200 multi

Western blotting was performed as described previously.⁴² (link) In brief, proteins were first extracted from cells or retinal tissues. Total protein (30 μg) was then loaded and separated on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes. Blots were incubated with a primary antibody against Mbd2 (ab188474; Abcam), Atf1 (11946-1-AP; Proteintech, Rosemont, IL), caspase3/cleaved caspase3 (#9662; CST, Danvers, MA), and β-tubulin (#10094-1-AP; Proteintech) at 4°C overnight. The following morning, membranes were washed and incubated with a species-specific horseradish peroxidase-conjugated secondary antibody, Goat Anti-Rabbit IgG (SA00001-2; Proteintech), at RT for 1 h. Positive binding was detected by an enhanced chemiluminescence kit (WBKLSO500; Millipore, Burlington, MA) and captured by a Tanon 5200 multi. Protein levels were normalized to β-tubulin and were compared with controls.

60 μg RNA in 20 μl DEPC-treated water was reacted with 50 μM DBCO-PEG4-biotin (stock solution in RNase-free DMSO) for 1 h at r.t. After extraction by TRIzol to remove the excess reagent, the resulted RNA was dissolved in 10 μl DEPC-treated water. 2 μl of RNA was loaded to a nylon membrane (hybond−N+) and crosslinked by irradiation with 254-nm UV light at 0.15 J/cm² twice using a UV crosslinker (CL-1000, UVP). The membrane was blocked by 10% SDS for 1 h and washed by TBST for three times. Then, the membrane was stained with streptavidin-HRP (0.5 μg/ml) for 1 h and followed by washing with 10% SDS, 5% SDS, 1% SDS, and three times of TBST for 5 min each. The blot was acquired using HRP substrate and peroxide solution (Millipore) on Tanon-5200 Multi.