Immunoblotting and cell-surface biotinylation

To detect Elkin1 variants at the plasma membrane, the cell surface fraction was biotinylated and subsequently isolated following established protocols (Tarradas et al., 2013). Briefly, HEK-293T P1KO cells attached to PLL-coated culture dishes were transiently transfected with plasmids encoding GFP-tagged Elkin1 variants. After 24 hr the cell surface fraction was labelled, on ice, with freshly prepared 2.5 mg/ml EZ-link Sulfo-NHS-LC-LC-biotin (21338, ThermoFisher Scientific) in DPBS containing Ca⁺⁺. After quenching with 100 mM glycine, cells were lysed as above using RIPA buffer containing protease inhibitor cocktail. A portion of this lysed sample was reserved as the ‘input’ sample. The biotinylated fraction was then isolated using NeutrAvidin Ultralink Resin (53150, ThermoFisher Scientific). After recovery from the NeutrAvidin beads, samples were prepared as for gel electrophoresis by mixing with Bolt LDS sample buffer and Bolt reducing agent (B0007 and B0009 respectively, ThermoFisher Scientific). Samples were separated on a 10% Bolt Bis-Tris Plus gel (NW00102BOX, ThermoFisher Scientific), transferred to a PVDF membrane and subjected to standard antibody detection. GFP-fusion proteins were detected using rabbit polyclonal anti-GFP (SAB4301138, Sigma-Aldrich, 1:1000) and HRP-linked anti-rabbit IgG (7074, Cell Signaling Technologies, 1:1000).