Cell surface protein binding assay

Cell surface binding assays were performed essentially as described^{27 (link), 29 (link)}. COS-7 cells were transfected with the expression vectors for candidate binding proteins and maintained for 24 h. The transfected COS-7 cells were washed with extracellular solution (ECS, containing 168 mM NaCl, 2.4 mM KCl, 20 mM HEPES (pH 7.4), 10 mM d-glucose, 2 mM CaCl₂ and 1.3 mM MgCl₂) that contained 100 μg ml^–1 bovine serum albumin (BSA) and then incubated with ECS and BSA containing 100 nM purified Fc-fusion protein for 1 h at room temperature. These cells were washed in ECS, fixed with parafix solution, incubated with blocking solution followed by rabbit anti-HA without permeabilization and then immunostained with Alexa488-conjugated goat anti-rabbit IgG (H+L) (1:500; Invitrogen) and Alexa594-conjugated donkey anti-human IgG (H+L) (1:500; Jackson ImmunoResearch) to label surface HA and bound Fc proteins, respectively. Images were acquired on a Leica DM6000 fluorescent microscope with a 40 × 0.75 NA dry objective and a Hamamatsu cooled CCD camera using Volocity software (Perkin Elmer).