Beta lactose

Nitrocellulose strips were blocked in a solution containing 0.3% casein (from 6% stock), 5 mM borate (ThermoFisher, Wattham, MA, 28341), 2% w/v beta lactose (Sigma, St Louis, MO, L3750) and 2% w/v sucrose (Sigma, St Louis, MO, S7903).
To block, striped nitrocellulose was placed with the long edge submerged in the blocking solution. The solution was allowed to wick until the nitrocellulose was fully wet, at which point it was submerged fully in the blocking solution. The nitrocellulose remained in the blocking solution for 15 minutes, with continuous rocking. Finally, the nitrocellulose was removed from the blocking solution and placed in a 25°C forced-air oven for 30 minutes or until dry. It was then immediately moved to an argon-purged desiccator for storage.

Soluble 4-1BB levels were measured as earlier publish (18 (link)). The galectin+4-1BB-binding assay were coated with either rhGal-3 or rhGal-9 (R&D Systems, USA) at 1 μg/ml with, or without, 0.1 M Beta-lactose (Sigma, L3750-100G) followed by addition of rh4-1BB 1 μg/ml (R&D Systems, USA). Finally, the inhibition-binding assays were evaluated by adding a biotinylated anti-4-1BB antibody 0.5 μg/ml (R&D Systems, USA) followed by Streptavidin HRP. The inhibition assays were incubating O.N at 4°C.