Fig. 3. Internalisation and half-life of LOX-1. Serum-starved LOX-1-FLAG transfected HeLa cells were incubated with or without saturating OxLDL levels for 1 hour and biotinylated as described in Materials and Methods. (A) Cells were incubated at 37°C for 0, 15 or 30 minutes before cell surface biotin cleavage followed by cell lysis. (B) Biotinylated cells were incubated at 37°C for different time periods before lysis. Biotinylated proteins were isolated and analysed by western blotting with purified anti-LOX-1 antibodies. The blot in A was reprobed with mouse anti-TfR (control). The amount of biotinylated LOX-1 was quantified using densitometry (mean ± s.e.m. of three experiments) and compared with total cell surface LOX-1 (t=0 minutes) to quantify percentage of LOX-1 internalised (A) or remaining (B).