Fig. 4

Silencing expression of the EFR3A gene in the Hela cell line induces changes in lateral membrane organization. Stable cell lines were obtained using EFR3A shRNA lentiviral particles or “scrambled” shRNA lentiviral particles as described in the Methods section. A Cell extracts of control or transduced cell lines were submitted to SDS-PAGE and western blotting probed with anti-EFR3A antibodies. GAPDH visualization was used as a loading control. B Quantitation of the EFR3A fractions shown in A. C Marked decrease of EFR3A level in cells is accompanied by a substantial reduction of EGFR in the DRM fraction. D Examples of FLIM images of the cell plasma membrane and E GPMVs derived from EFR3A KnD and “scrambled” cells. F and G Quantitation of FLIM data of EFR3A KnD and “scrambled” cells and corresponding GPMVs. For FLIM measurements of the fluorescence lifetime of the membrane-order sensitive probe, di-4 ANEPPDHQ (Invitrogen) control “scrambled” and EFR3A KnD HeLa cells were grown in LabTek chambers in DMEM medium with 10% FBS. After 24 h cells were washed twice and stained with a 2 μM di-4 probe in DMEM medium for 5 min. Cells were washed twice in HBSS buffer with 10 mM HEPES pH 7.4 and measurements were performed. Giant plasma membrane vesicles (GPMV) were generated from EFR3A KnD and “scrambled” cells as mentioned in the Methods section and stained with di-4 (2 μM) as above. The lifetime values of wild-type (WT) cells did not differ from “scrambled”, so we did not include them for clarity