Iseq00005

To detect protein expression in mouse materials, total proteins were extracted using WIP Tissue and Cell lysis solution (BioChip, 110000) according to the manufacturer’s protocol. Electrophoresis was performed with 50 μg total proteins separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, ISEQ00005). The membranes were incubated overnight at 4 °C with the appropriate primary antibodies listed below: anti-RDX (78 kDa, 1:500, ab52495, Abcam), anti-p-ERM (78 kDa, 1:500, mAb#3726, Cell Signaling Technologies). The appropriate secondary antibody (ZB-2301, ZB-2305 from ZSGB-BIO) was diluted 1:5000 in TBST (TBS plus 0.5% Tween 20). Tubulin (1:5000, Beyotime, AF5012) was used as an internal control. The membranes were visualized using the SuperSignal chemiluminescent detection system (Thermo Scientific, 32109). Original blots can be found in the Source data file.

RPE/choroids and neural retinas from WT and Cas9 mice were lysed in 1x RIPA buffer (Thermo Scientific 89900, Rockford, IL, USA) containing protease (Thermo Scientific, A32963), and phosphatase (Thermo Scientific A32957, Rockford, IL, USA) inhibitor cocktails. Tissue lysates were quantified using the Rapid Gold BCA Protein Assay Kit (Pierce A53226, Rockford, IL, USA), and 25 µg protein was loaded onto 4–20% Tris-Glycine gels (Thermo Scientific XP04200BOX, Rockford, IL, USA). Proteins were transferred onto PVDF membrane (Millipore ISEQ00005, Burlington, MA, USA), blocked with 5% skimmed milk in phosphate-buffered saline (PBS) for 1 h, and incubated overnight at 4 °C in an orbital shaker using the following primary antibodies: anti-Cas9 (1:1000; Cell Signaling Technology 19526, Danvers, MA, USA) or anti-β-actin (1:1000; Cell Signaling Technology 4970, Danvers, MA, USA). Membranes were washed thrice with PBS-T and stained with HRP-conjugated secondary antibodies (1:5000; Invitrogen G21234, Carlsbad, CA, USA) for 2 h at room temperature. Stained membranes were washed thrice in PBS-T, and then developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific 34580, Rockford, IL, USA) and imaged using a gel imager (Azure Biosystems 300Q, Dublin, CA, USA).