RAB10-Galectin-3 Binding Assay

The RAB10/Gal-3 cell-free binding assay was performed as reported^{14 (link)}. Briefly, 96-well plates were coated with purified human Gal-3 (Biolegend, 0.5 µg in 100 µl), incubated at 4 °C overnight, and then blocked with 50 mg/mL bovine serum albumin (BSA) for 90 min at 30 °C. After washing, recombinant human RAB10 (Prospec PRO-1361, from 0.25 to 0.5 µg/well) and MCP were combined and added for a total volume of 100 µl, then incubated for 4 h at 30 °C. Wells were washed, fixed with 2% PFA in PBS for 15 min at room temperature, washed, and then incubated with rabbit monoclonal RAB10 antibody (Abcam, 0.5 mg/mL diluted 1:100) for 1 h at RT. Wells were washed again and incubated with the secondary antibody (Life Technologies A11034, AF488 goat anti-rabbit IgG or A21206, AF488 donkey anti-rabbit IgG, both diluted 1:200) for 1 h at RT. Wells were washed three times and fluorescence read using a Cytation3 reader (BioTek) (ex.: 485 nm, em.: 538 nm) to quantify the binding of RAB10 to Gal-3.