Fig. 4. A PtdIns(3)P probe is redirected from EEA1-positive regions into Hrs-containing microdomains through clathrin binding. (A) Schematic representation of the 2xFYVE-Hrs CT chimeric constructs, with and without the clathrin-box motif. HeLa cells were transiently co-transfected with Rab5^Q79L and (B) myc-2xFYVE-Hrs CT or (D) myc-2xFYVE-Hrs CT_CB, and stained with anti-myc and anti-EEA1 antibodies for immunofluorescence confocal microscopy. Alternatively, cells were triple transfected with Rab5^Q79L and (C) myc-2xFYVE-Hrs CT and YFP-Hrs or (E) myc-2xFYVE-Hrs CT_CB and YFP-Hrs, and stained with anti-myc antibodies. Insets show two different enlarged endosomes from each transfection. Yellow indicates colocalisation. Bar, 5 µm. The level of colocalisation of the different proteins on endosome membranes was quantified as described in Materials and Methods (F). Error bars give the mean + s.e.m. myc-2xFYVE-Hrs CT + EEA1, n=19; myc-2xFYVE-Hrs CT + YFP-Hrs, n=27; myc-2xFYVE-Hrs CT_CB + EEA1, n=27; 2xFYVE-Hrs CT_CB + YFP-Hrs, n=29.