PLAP endocytosis assay

After cholesterol depletion or sterol substitution, the plates containing the cells were placed on ice and washed twice with 2 ml cold 1× PBS (i.e. placed on ice). Mouse monoclonal anti-PLAP IgG was prepared at 20 µg/ml in HBSS containing 3% w/v BSA. Each plate was incubated with 100 µl of anti-PLAP antibody solution at 4°C for 15 min, then placed on ice, and washed twice with 2 ml cold 1× PBS. Next, 100 µl of AF488-conjugated goat anti-mouse-IgG antibody [prepared at 40 µg/ml in HBSS with 3% (w/v) BSA] was added to the cells and incubated for 15 min at 4°C. Then the plates were again washed twice with 2 ml cold 1× PBS. Pre-warmed 3% (w/v) BSA in HBSS was then added, and plates were incubated at 37°C for 20 min to induce internalization of PLAP. Cells were placed on ice to stop internalization and washed twice with 2 ml cold 1× PBS. By addition of cold PBS to the cells and incubating them on ice instead of 37°C, we also prepared an untreated control in which internalization was not induced because the cells were kept on ice. Cells were moved into a cool room (16–18°C) and acid-washed three times (incubating in acid for 3 min each time) with 2 ml of 100 mM citric acid and 140 mM NaCl, adjusted to pH 1.75 with HCl. Fig. S5 shows the acid washing worked properly, with over 90% of fluorescence on the cell surface removed. Then cells were washed twice with 2 ml cold 1× PBS and, after removal of the washing solution, fixed with 2 ml of 3% (w/v) PFA in PBS for 10 min on ice followed by 20 min at room temperature. After fixation, cells were washed three times with 2 ml 1×PBS at room temperature. Cellular plasma membrane staining was performed with 100 µl CellMask solution diluted to 4 µl/ml in HBSS with 3% (w/v) BSA for 2–5 min at room temperature. After washing with 2 ml 1× PBS at room temperature, coverslips were taken from the dishes and mounted on slides using VECTASHIELD® mounting medium.

The minimal fluorescence present after acid washing when cells are not exposed to temperatures over 18°C, could be due to the residual baseline endocytosis plus a small amount of autofluorescence. To correct for this, we only consider the increase over the background value of fluorescence (that in these low temperature samples) as representing sterol-supported endocytosis.