Bs 7565r

Recombinant human GYPC protein (0, 0.5, and 5 μg, Prospec-Tany Technogene) diluted in phosphate-buffered saline (PBS) was coated onto high-protein-binding 96-well plates (Corning, Inc., Corning, NY, USA) overnight at room temperature. After three washes with PBS containing 0.05% Tween 20 (PBS-T), blocking buffer containing 1% bovine serum albumin (BSA) in PBS-T was added to the 96-well plates, and the plates were incubated for 2 h at room temperature. Recombinant human ORM2 (Prospec-Tany Technogene) or BSA (Bovogen, Keilor East, VIC, AUS) at concentrations of 0, 5, 25, and 125 μg was then added to the plate and incubated overnight at 4 °C. After three washes with PBS-T, rabbit anti-ORM2 Ab (bs-7565r, Bioss) was added to the plates, and the reactions were allowed to proceed for 2 h at room temperature. After washing three times with PBS-T, goat anti-rabbit IgG Ab conjugated with HRP (21040 s, Thermo Fisher Scientific) was added as the secondary Ab, and the reaction was again allowed to proceed for 1 h at room temperature. After three additional washes with PBS-T, TMB solution was added to induce a color reaction, which was then stopped with H₂SO₄ (sulfuric acid) solution. Finally, the optical densities were read at a wavelength of 450 nm.

RA-FLSs (4 × 10³ cells per well) were seeded in eight-well chambers (Thermo Fisher Scientific) and grown to 80% confluence for 24 h. Recombinant ORM2 (1 μg/mL; Prospec-Tany Technogene) was added to each well and incubated at 37 °C for 1 h. The cells were then fixed with formaldehyde, permeabilized with 0.1% Triton X-100 for 3 min at room temperature, and incubated with an anti-ORM2 Ab (bs-7565R, 1:100; Bioss, Woburn, MA, USA) and/or an anti-GYPC Ab (sc-59183, 1:100; Santa Cruz Biotechnology) for 1 h at 37 °C. These cells were then stained with a Duolink In Situ Orange Starter Kit Mouse/Rabbit (Sigma‒Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Approximately 50–100 cells per area (at a magnification of 100×) were observed, and more than 5 randomly selected areas were subjected to analysis.