Everyblot

Conditioned culture medium was mixed with Laemmli buffer and proteins were separated on a 4–15% Mini-PROTEAN TGX stain-free protein gel (Bio-Rad) using a tris-glycine-SDS buffer (Bio-Rad). Proteins were then transferred to PVDF membranes, which were subsequently blocked (EveryBlot, Bio-Rad) and then probed with a home-made rabbit anti-DS-epi1 (1 μg ml^–1, antigen: amino acids 509–520) in blocking buffer (EveryBlot, Bio-Rad). A goat anti-rabbit IgG (H + L)-HRP conjugate (Bio-Rad, 1706515) was used together with a clarity western ECL substrate (Bio-Rad) to develop the blot, which was imaged using a ChemiDoc imaging system (Bio-Rad).

Polarized ARPE-19 monolayers were generated and treated in 12-well plates. Proteins were isolated with RIPA lysis buffer (Santa Cruz Biotechnology, USA), separated by SDS page using 10% (receptor analysis) or AnykD (LC3B analysis) TGX stain-free gels (Bio-Rad) and transferred to a PVDF membrane (Amersham Hybond, GE Healthcare, IL, USA) by wet electroblotting (Bio-Rad). Protein expression was normalized to the amount of total loaded protein (Image Lab 6.0.1, Bio-Rad). For detection of CysLTR1, the membrane was blocked with EveryBlot (Bio-Rad) for 10 min at room temperature and incubated overnight with an anti-CysLT1 antibody (ab151484, Abcam) diluted 1:500 in EveryBlot blocking solution at room temperature. CysLTR1 was visualized using an anti-rabbit antibody conjugated to HRP (Agilent, CA, USA) and Clarity Western ECL Substrate (Bio-Rad). For detection of LC3-I and LC3-II, the membrane was blocked with 5% milk powder for 1 h at room temperature and incubated overnight with a recombinant anti-LC3B antibody [EPR18709] (ab192890, Abcam, UK) diluted 1:1000 in blocking solution at 4 °C. LC3B was visualized using an anti-rabbit antibody conjugated to HRP and Clarity Western ECL Substrate. Chemiluminescence was detected using the ChemiDoc XRS + system (Bio-Rad). Full-length blots are presented in the supplementary material.

Sectioned membranes of ALI cultures were added to RIPA (Radioimmunoprecipitation assay) buffer (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail on ice for 1 h and centrifuged at 12,000×g for 10 min at 4 °C. Total protein extracted was quantified using BCA assay kit (Pierce, Thermo Fisher Sci, USA) on the spectrophotometer (BioTek, USA) at 450 nm. Equal concentrations (20 µg) of protein were used for SDS-PAGE based protein separation on 4–12% gels (Bio-Rad) and transferred on a PVDF membrane (Millipore, Sigma, USA). Membranes were blocked using Everyblot (BioRad, USA) for 20 min at room temperature, incubated with primary rabbit raised polyclonal antibody against ANO1 (1:1000; Invitrogen, Thermo Fisher Scientific, USA), mouse monoclonal antibody against β-actin (1:500; Sigma-Aldrich, USA) overnight at 4 °C. Membranes were washed with TBST for 5 min and incubated with respective HRP-conjugated anti-rabbit and anti-mouse secondary antibody (Sigma-Aldrich, USA) for 2 h at room temperature. Protein detection was done using the ECL detection system (Thermo Fisher Sci, USA) and relative quantification for band intensity was done using ImageJ software.