Rabbit anti p62

Cells grown in the presence or absence of 20 μM Chloroquine (C-6628 SIGMA-ALDRICH, St. Louis, MO, USA) for 4 hrs were lysed in an extraction buffer consisting of 25 mM Tris, 50 mM NaCl, 2% Igepal, 0.2% SDS and 2 mg/ml protease inhibitor 18 (Complete, Roche Molecular Diagnostics, pH 7.4). Thirty micrograms of total protein were separated by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF-FL) membranes (Millipore). Membranes were incubated overnight with the primary antibody at 4 °C, rabbit anti-LC3 (1:1000, MBL PD014, Nagoya, Japan), rabbit anti-p62 (1:500, CELL SIGNALING 5114S, Beverly, MA, USA), mouse anti-TUBULIN (1:10000, CELL SIGNALING 3873, Beverly, MA, USA). Following three washes with TTBS secondary antibody IRDye^® 680RD goat anti-rabbit (925-68071, LI-COR) or IRDye^® 800CW goat anti-mouse (925-32210, LI-COR) was applied at 1:10,000 dilution in TTBS. Membranes were scanned and analyzed using an Odyssey® IR scanner and Odyssey® Image Studio software 5.2.5.

The following primary antibodies were employed for either immunofluorescence or western blotting: mouse anti-ARH-I (for IF 1:250, for WB 1:1000; cod. ab45768; Abcam, Cambridge, UK), mouse anti-p21 (for IF 1:100, for WB 1:200; cod. sc-817; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-LC3 (1:2000; cod. L7543; Sigma-Aldrich), rabbit anti-p62 (1:500; cod. 8025; Cell Signaling, Danvers, MA, USA), mouse anti-β-actin (1:2000; cod. A5441; Sigma-Aldrich), mouse anti-BECLIN-1 (1:500; cod. 612112; BD Biosciences, Franklin Lakes, NJ, USA), goat anti-BECLIN-1 (1:250; cod. sc-10086; Santa Cruz Biotechnology), rabbit anti-VPS34 (1:500; cod. 4263; Cell signaling), mouse anti-STAT3 (1:500; cod. 9139; Cell Signaling), rabbit anti-phospho (Tyr705) STAT3 (1:1000; cod. 9145; Cell Signaling), rabbit anti-Ki-67 (1:100; cod. HPA001164; Sigma-Aldrich), mouse anti-BCL-2 (1:500; cod. 15071; Cell Signaling), mouse anti-IL-6R (1:1000; cod. AHR0061; Invitrogen, Waltham, MA, USA), rabbit anti-cyclin D1 (1:500; cod. 2978; Cell Signaling), rabbit anti-p38 (1:500; cod. 9212; Cell Signaling), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma Aldrich), and mouse anti-ERK1/2 (1:500; cod. 05-1152; Millipore, Burlington, MA, USA).

The proteins were harvested and lysed in RIPA buffer. Equal amounts of protein extracts (30 μg) were loaded per lane and resolved by SDS/PAGE. Subsequently, polypeptides were separated and transferred to PVDF membranes. The membranes were blocked and then treated overnight with rabbit anti-Beclin-1 (1 : 1000, Cell Signaling Technology), rabbit anti-Atg7 (1 : 1000, Cell Signaling Technology), rabbit anti-Atg5 (1 : 1000, Cell Signaling Technology), rabbit anti-P62 (1 : 1000, Cell Signaling Technology), rabbit anti-LC3II/I (1 : 1000, Sigma), and mouse anti-β-actin (1 : 1000, Beyotime Institute of Biochemistry) antibodies, respectively. After being washed with TBST three times, the membranes were incubated using the corresponding HRP-conjugated secondary antibodies (1 : 1000, Beyotime Institute of Biochemistry) with blocking solution at room temperature for 2 h. Finally, the expression levels of protein were measured by enhanced chemiluminescence kit (Millipore). The protein bands were normalized by β-actin and quantified as the ratio of the optical density.

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.

Cycloheximide (catalog no. 14126) was purchased from Cayman Chemical (MI, USA), and MG-132 (catalog no. tlrl-mg132) was obtained from InvivoGen (CA, USA). Rabbit anti-LC3 (catalog no. 4108S), rabbit anti-Beclin1 (catalog no. 3738S), and rabbit anti-p62 (catalog no. 8025S) were purchased from Cell Signaling Technologies (MA, USA). Rabbit anti-actin (catalog no. A300-485A) was purchased from Bethyl Laboratories (TX, USA). Mouse anti-Flag (catalog no. F1804) and mouse anti-HA (catalog no. 26183) antibodies were obtained from Sigma-Aldrich (MO, USA) and ThermoFisher Scientific (MA, USA), respectively. Fluorescein isothiocyanate (FITC) goat anti-rabbit IgG (catalog no. 111-095-003) and Alexa Fluor 594 donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (PA, USA). Anti-ISG15 antibody was generated as described previously (46 (link)). Interferon beta (catalog no. 10704-HNAS) was purchased from Sino Biological US, Inc. (PA, USA).