Rabbit anti lc3 2

Cell lysates were obtained after incubation in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 8.0) supplemented with complete protease inhibitor and resuspended in Laemmli buffer. Crude lysates were boiled for 5 m and then kept on ice. Proteins were separated by SDS-PAGE, transferred to PVDF membrane and incubated with 1 : 1000 rabbit anti-STING (Cell Signaling Technology, #13647S), 1 : 1000 rabbit anti-cGAS (Cell Signaling Technology, #15102S), 1 : 1000 rabbit anti-pSTING 1 : 1000 (Cell Signaling Technology, #85735S), rabbit anti-LC3-II (Cell Signaling Technology, #2775S) or 1 : 3000 mouse anti-LAMP1 (eBioscience, eBioH4A3) followed by 1 : 10 000 HRP-conjugated anti-rabbit or anti-mouse. Blots were developed using ECL™ Prime Western Blotting Detection Reagents.

Expression of LC3-II and p62 proteins was determined by Western blotting (6 eyes per group). The proteins were extracted from the corneal and conjunctival tissues using a lysis buffer. The lysates were centrifuged at 25,200× g for 10 min at 4 °C. The proteins (40 μg) of the samples were loaded by 10% SDS-PAGE for 30 min at 80 V. Following gel loading, the samples were transferred from gel to the membranes and blocked using skim milk (non-fat milk) for 60 min at room temperature. The membranes included rabbit anti-LC3-II (catalog no. ab192890), or rabbit anti-p62 (catalog no. ab56416; primary antibodies obtained from Cell Signaling Technology, Beverly, MA, USA) in 1 × TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20) overnight at 4 °C. The next day, the membranes were washed three times with 1 × TBST buffer for 5 min and incubated with secondary antibodies in 1 × TBST for 60 min at room temperature. After incubating, the samples were washed three times with TBST for 5 min. The images of immunoreactive bands were captured using an enhanced chemiluminescence system (ECL Blotting Analysis System; Amersham, Arlington Heights, IL, USA). Anti-β-actin was used as an inner control.

Western blotting was performed as described in [24] (link). Blots were incubated with either mouse anti-β-glucocerebrosidase (overnight at 4 °C, diluted 1:500, EMD Millipore), mouse anti-LAMP1 (1 h at RT, diluted 1:500, Santa Cruz Biotechnology) or rabbit-anti-LC3II (overnight at 4 °C, diluted 1:1000, Cell Signalling) primary antibodies. Blots were stripped and re-probed with a goat anti-actin (1 h at RT, diluted 1:500, Santa Cruz Biotechnology) primary antibody. Anti-mouse (Santa Cruz Biotechnology), anti-rabbit (Bio-Rad) or anti-goat (Santa Cruz Biotechnology) IgG conjugated to horse-radish peroxidase were used as the secondary antibodies (1 h at RT, 1:2000).