The ROs were pooled (10) and lysed in radioimmunoprecipitation assay protein lysis buffer (Abcam) with protease inhibitor cocktail (Roche, Basel, Switzerland) and homogenized using a 25G needle. The protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and the plate absorbance was read in the SpectraMAX plate reader (Molecular Devices, San Jose, CA, USA) at 562 nm. The samples (22.5–85 μg) were loaded on 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad), ran for the first 30 min at 70 V and the next 4 h at 100 V in Tris-Glycine SDS buffer. The gels were transferred to PVDF membranes (Merck KGaA) previously activated with methanol, in 1× Tris-Glycine buffer and 20% methanol at 70 mV overnight at 4°C. The membranes were rinsed in PBS-0.1% Tween, blocked in Pure Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA) for 2 h and incubated with an anti-ABCA4 clone 5B4 (1:1,000; Merck KGaA) and anti-vinculin (1:5,000; Abcam) at 4°C overnight. The membranes were washed with PBS-0.1% Tween and incubated with Goat Anti-Mouse IRDye 800 and Goat Anti-Rabbit IRDye 680 (1:5000; Li-COR Biosciences) for 1.5 h in the dark. The membranes were washed with PBS-0.1% Tween and scanned wet in the Odyssey IR system (Li-COR Biosciences). The intensity of the detected bands was quantified using FIJI ImageJ 1.53c, and the samples were normalized to the wild-type sample. The mean percentage of the detected ABCA4 protein was statistically analyzed using GraphPad Prism 9, with ordinary one-way ANOVA test followed by Dunnet’s multiple comparison test.
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