Protein partitioning measurements in giant plasma membrane vesicles (GPMVs)

Cell membranes were stained with 5 μg/ml of Texas Red DHPE or Annexin V 647 (ThermoFisher), respectively, red or far-red fluorescent lipid dyes that strongly partition to disordered phases (Baumgart et al., 2007; Klymchenko and Kreder, 2014; Stone et al., 2017). Following staining, GPMVs were isolated as described (Sezgin et al., 2012) from U2OS stable cells lines expressing the protein of interest (LAT results were obtained from transient co-transfections). Briefly, GPMV formation was induced by 2 mM N-ethylmaleimide (NEM) in hypotonic buffer containing 100 mM NaCl, 10 mM HEPES, and 2 mM CaCl₂, pH 7.4. To quantify partitioning, GPMVs were observed on an inverted epifluorescence microscope (Nikon) at 4°C after treatment with 200 µM DCA to stabilize phase separation; this treatment has been previously demonstrated not to affect raft affinity of various proteins (Castello-Serrano et al., 2020). The partition coefficient (K_p,raft) for each protein was calculated from fluorescence intensity of the construct in the raft and non-raft phase for >10 vesicles/trial, with multiple independent experiments (n = 3) for each construct.