Las 3000 machine

Nymphal (5^th instar) or adult salivary proteins (50 µg protein in a 2D well) were separated by SDS-PAGE as described above and transferred onto a nitrocellulose membrane at 25 V for 30 min using the Pierce Fast Semi-dry Blotter (Thermo Scientific). After the transfer, the membrane was blocked with 5% skimmed milk in PBS overnight at 4°C, washed three times in PBST and incubated with guinea pig sera diluted 1∶100 in PBST with 5% skimmed milk for 1 h at room temperature using the Mini-Protean II Multiscreen apparatus (Bio-Rad). Following repeated washing steps, the Western blot membrane was incubated for 1 h at room temperature with HRP conjugated rabbit anti-guinea pig IgG secondary antibodies (Sigma-Aldrich) diluted 1∶10,000 in PBST with 5% dried skimmed milk or HRP goat anti-guinea pig IgM secondary antibodies (Immunology Consultants Laboratory), diluted 1∶5,000 in PBST with 5% dried skimmed milk. The proteins were visualized using the Pierce ECL Western Blotting Substrate (Thermo Scientific) and digitized using the LAS-3000 machine (Fujifilm).

Corneal and conjunctival tissues were homogenized in RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0, 1× protease inhibitors, and 1 mM phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 14,000× g for 20 min, and total protein concentrations were determined with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were labeled with mouse anti-TNF-α (Santa Cruz Biotechnology, Paso Robles, CA, USA) and mouse anti-β-actin antibodies (Sigma, St. Louis, MO, USA). The immunoreactive bands were detected using chemiluminescence detection reagents (Pierce, Rockford, IL, USA), and the density of the bands of interest was measured using a LAS-3000 machine (Fujifilm, Tokyo, Japan). Anti-β-actin antibody served as a loading control.

15x10⁴ hMVECs (on 5 cm²) were washed with PBS and lysed in Laemmli Sample Buffer (Bio-Rad, Hercules, USA) with 5% β-mercaptoethanol. Protein was separated on an 8% SDS-polyacrylamide gel and electrophoretically transferred onto nitrocellulose membrane (Amersham, Uppsala, Sweden) in a buffer of 192 mM glycine, 25 mM Tris (pH 8.3) and 10% (v/v) methanol. The membranes were blocked with 5% (w/v) non-fat milk in 137 mM NaCl, 20 mM Tris (pH 7.6) and 0.1% Tween 20 (TBST) for 90 minutes, followed by overnight incubation at 4°C with the primary polyclonal antibodies (anti-HIF-1α 1:250 (Cayman chemical; 10006421), anti-HIF-2α 1:500 (Novus Biologicals; NB100-122)) in TBST + 5% non-fat milk. Subsequently, the blots were washed three times with TBST and incubated for 90 minutes at room temperature with horseradish-conjugated goat-anti-rabbit antibodies (1:5000) or horseradish-conjugated goat-anti-mouse antibodies (1:5000) (Dako, Darmstadt, Germany) in TBST + 5% nonfat milk as a conjugate. The bands were visualized with enhanced chemiluminescence (Sigma) on a LAS3000 machine (Fujifilm, Japan).