Mouse anti rpe65

Following treatment, cells were fixed with 4% paraformaldehyde for 10 min before permeabilization with 0.1% Triton X-100 in PBS for 10 min. Cells were then blocked for 1 h in normal goat or horse serum before overnight incubation at 4 °C with their respective primary antibodies; anti-NLRP3 (1:500; Abcam, Cambridge, UK), anti-cleaved caspase-1 (1:100, Invitrogen, Carlsbad, USA), anti-NLRP1 (1:500; Novus Biologicals, Littleton, CO, USA), or mouse anti-RPE65 (1:1000; Abcam, Cambridge, UK). Two 10 min washes in PBS followed, after which cells were incubated at room temperature for 2 h with their respective secondary antibodies; donkey anti-rabbit Alexa-488 (1:500; Abcam, UK), donkey anti-mouse Alexa-488 (1:500; Abcam, Cambridge, UK), or donkey anti-goat Cy3 (1:500; Invitrogen, Carlsbad, USA). Cells were then washed twice in PBS for 10 min. Cell nuclei were counterstained with DAPI (1:1000; Sigma-Aldrich, St Louis, MO, USA), and slides were mounted using a CitifluorTM antifade reagent.

RPE cells were cultured for 14 days and lysed by RIPA buffer (Beyotime, Shanghai, China) for 30 min. After centrifugation, total protein concentrations of all samples were quantified using a BCA Protein Quantification Kit (Vazyme) according to the manufacturer’s protocols. Equal amounts (30 μg) of proteins per lane were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to nitrocellulose membranes. After blocking with PBS containing 3% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies, including rabbit anti-PRPF6 (1:1000, Invitrogen), rabbit anti-CRALBP (1:1000, Proteintech), mouse anti-RPE65 (1:1000, Abcam), rabbit anti-tyrosinase (1:1000, Abcam), mouse/rabbit anti-GAPDH (1:2000, Arigo) and mouse anti-β-Actin (1:2000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Then, membranes were incubated with goat anti-rabbit IRDye 680RD secondary antibodies or goat anti-mouse IRDye 800 CW secondary antibodies (1:10,000, LI-COR Bioscience, Lincoln, NE, USA) for 1 h at room temperature. The bands were imaged on the Odyssey Fc Imaging System (LI-COR Bioscience), and the results were analyzed by Fiji/Image J software (National Institutes of Health, Bethesda, MD, USA).

RPE cells were cultured for 14 days and washed two times with PBS. After fixing with 4% paraformaldehyde (PFA), cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in PBS for 1 h at room temperature. Then, cells were incubated overnight at 4 °C with primary antibodies, including mouse anti-ZO-1 (1:100, Invitrogen, Carlsbad, CA, USA), rabbit anti-MITF (1:200, Invitrogen), mouse anti-RPE65 (1:50, Abcam, Cambridge, UK), rabbit anti-CRALBP (1:100, Proteintech, Rosemont, IL, USA), rabbit anti-tyrosinase (1:100, Abcam), rabbit anti-EZRIN (1:200, GeneTex, Irvine, CA, USA), mouse anti-Na⁺/K⁺-ATPase (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-Collagen IV (1:100, Abcam) (Appendix B, Table A1) followed by washing three times in PBS and incubation with secondary goat anti-mouse/rabbit Alexa Fluor 594/488 antibody (1:1000, Invitrogen) for 1 h at room temperature. After washing three times with PBS, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, Beijing, China) for 10 min. Immunofluorescence images were captured using a laser scanning confocal microscope (LSM800; Zeiss, Thornwood, Germany).

Calcium and magnesium free PBS (PBS⁻) was purchased from Gibco (#10010-023). Triton X-100 was obtained from Sigma-Aldrich (#T8787). Hoechst 33258 (#H3569) and AlexaFluor 568-conjugated Phalloidin (#A12380) were from Invitrogen. Antibodies used were as follows: Mouse anti-RPE65 (#ab78036 [clone 401.8B11.3D9]; Abcam, Cambridge, MA), mouse anti-Cytokeratin (#M0821 [clone MNF116]; Dako A/S, Glostrup, Denmark), rabbit anti-ZO1 (mid) (#40-2200; ThermoFisher Scientific, Waltham, MA), mouse anti-Syntenin-1 (#ab131190 [clone 3D9-G9-H4]; Abcam), mouse anti-TSG101 (#612696; BD Biosciences, San Jose, CA), rabbit anti-SLC39A12 (#ab106570; Abcam), rabbit anti-Calreticulin (#12238 [clone D3E6]; Cell Signaling Technologies, Danvers, MA), mouse anti-BEST1 (#NB300-164, [clone E6-6]; Novus Biologicals, Littleton, CO), mouse anti-Na⁺/K⁺-ATPase alpha (#sc-58628 [clone M7-PB-E9]; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-CD36 (#ab78054; Abcam), AlexaFluor 488-conjugated donkey-anti-rabbit IgG (#A21206, Invitrogen), AlexaFluor 488-conjugated donkey-anti-mouse IgG (#A21202, Invitrogen), AlexaFluor 568-conjugated donkey-anti-rabbit IgG (#A10042; Invitrogen), HRP-conjugated donkey-anti-mouse IgG (#715-035-150, Jackson ImmunoResearch Laboratories, West Grove, PA), and HRP-conjugated donkey-anti-rabbit IgG (#711-035-152, Jackson ImmunoResearch Laboratories).