Ab188474

Retina tissues and cells were lysed using a lysis buffer (125 mM HCl [pH 6.8], 4% SDS, 20% glycerol, urea 0.36 g/mL, 20 mM DTT, 1% proteinase inhibitor cocktail), and the proteins were resuspended in SDS-PAGE loading buffer. Equal amounts of samples were loaded in each lane. The proteins were separated in 12% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were then probed with primary antibodies against Mbd2 (GenBank: 17191) (#ab188474; Abcam, Cambridge, MA, US), caspase3/cleaved caspase3 (#9662; CST, Danvers, MA, US), Traf3 (GenBank: 22031) (#ab36988; Abcam), and β-tubulin (#10094-1-AP; Proteintech, Rosemont, IL, US), followed by incubation with species-specific horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, SA00001-2; Proteintech), and visualized by enhanced chemiluminescence (Millipore; WBKLSO500). Finally, the images were captured by Tanon 5200 Multi. The target proteins were quantified by Photoshop software and normalized by internal control β-tubulin to get relative expression levels.

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.