2.4. Surface biotinylation assay

The surface biotinylation assay was performed with Pierce Cell Surface Biotinylation and Isolation Kit (Thermofisher Scientific, A44390) with minor modification. In brief, 1.5 μg of WT, 27A, and 27B plasmids were transfected into 1.5 million HEK293FT cells cultured on 6-well plates. 48 hours post-transfection, the cells were labelled with EZ-Link Sulfo-NHS-SS-Biotin and subsequently lysed with 300 μL NP40 buffer containing 150 mM sodium chloride 1.0% Triton X-100 and 50 mM Tris, pH 8.0 supplemented with Mini, EDTA-free Protease Inhibitor Cocktail (Roche). 30 μL of the clarified lysate was saved as total lysate whereas the remaining lysate was treated to 100 μL of NeutrAvidin Agarose beads to enrich for biotin-labelled surface fraction. The beads were subsequently washed 4 times with 1 mL NP40 buffer and denatured at 95°C with 90 μL of NP40 buffer supplemented with 6x loading buffer before SDS-PAGE and Western blot. Transferrin receptor (TFR) was used a loading control for surface protein detected with TFR antibody (Thermofisher Scientific, 136800) at 1:1000 dilution.