Cocktail of protein inhibitors

For protein extraction, the samples control (n = 2) and hypoxic (n = 2) retinas were homogenized with RIPA Lysis Buffer (Sigma-Aldrich, St. Louis, MO, USA) and a cocktail of protein inhibitors (Sigma-Aldrich), and sonicated with a Sonopuls Ultrasonic homogenizers (Bandelin). The protein concentration was determined by the Bicinchoninic acid assay. Samples were mixed with the Morris formulation of the SDS-PAGE Sample Buffer (2.5% SDS, 10% glycerol, 5% β-mercaptoethanol) and denatured for 10 min at 98 °C and separated on 10% SDS-polyacrylamide gels (20 and 40 µg/lane). For immunoblotting, proteins were transferred to a nitrocellulose membrane, blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies: rabbit anti-RBPMS antibody, and in parallel rabbit anti-β-actin antibody was used as internal control overnight at 4 °C. After incubation with the secondary antibody, the blotting membrane was developed with Luminata™ Crescendo Western HRP Substrate (Millipore, Burlington, MA, USA) in a G: BOX (Syngene, Cambridge, UK) and the images were obtained with the GeneSnap Software and analyzed with ImageJ software (version 1.4.3.67) (NIH, Bethesda, MD, USA).

Larvae and adult antigens were prepared as described above in order to detect stage-specific antibody levels. One million 3rd stage (L3) larvae of H. contortus were used to prepare somatic antigens of larvae^lH. contortus. Adult Somatic Antigens were prepared with adult worms of H. contortus collected from donor sheep with a primary infection of 7000 L3 H. contortus. Larvae or adult worms were exposed to three cycles of 20 min of freeze-thawing (from −80 °C to room temperature) in PBS with a cocktail of protein inhibitors (Sigma-Aldrich, St. Louis, MO), homogenized mechanically and followed by ultrasound disruption at 4 °C. The homogenate was then centrifuged at 10,000 g for 30 min at 4 °C and the supernatant was stored at −80 °C until use. Protein concentration of larval and adult lysates was determined using the Bicinchoninic Acid protein assay procedure.