Fig. 6. Clathrin and AP2-independent uptake of OxLDL by LOX-1. Following RNAi, LOX-1 was expressed in transfected HeLa cells and labelled transferrin and OxLDL uptake was monitored (see Materials and Methods) by microscopy (A) and quantified (C). (A) HeLa cells subjected to RNAi through mock treatment (mock), a control scrambled siRNA duplex (scrambled), a siRNA duplex specific for the clathrin heavy chain (CHC17) or a siRNA duplex specific for the µ2 subunit of the AP2 adaptor complex (µ2) on cells expressing LOX-1-FLAG. After 12 hours, cells were incubated with Alexa Fluor 488-transferrin and DiI-OxLDL for 15 minutes followed by 30 minute chase and then fixation and confocal laser-scanning microscopy. Arrows (left hand panels) indicate plasma membrane transferrin accumulation in cells (^*) where clathrin or AP2-mediated uptake is inhibited. In right panels, transverse z-axis sections are also shown to visualise intracellular staining; small arrows denote endosomes containing labelled OxLDL. Scale bars: 10 µm. (B) Western blotting to demonstrate depletion of endogenous protein levels after RNAi treatment using CHC17 siRNA (lane 1), µ2 siRNA (lane 2), scrambled siRNA (lane 3) and mock-transfected cells (lane 4). (C) Quantification of uptake of labelled transferrin and OxLDL ligands in LOX-1-transfected HeLa cells (n=30, error bars indicate s.e.m.) was carried out as described in Materials and Methods.