Fig. 5
“Rescue expression” of EFR3A in KnD HeLa cells. A Western blotting of total protein extracts of EFR3A KnD HeLa cells transfected with p3xEFLAG-CMV10 EFR3A (lane 1) and control p3xEFLAG-CMV10 plasmids (lane 2). Anti-FLAG antibodies (Merck) were used as primary antibodies. B Representative di-4 FLIM images of control, “scrambled” cell (left), EFR3A KnD cell transfected with an empty vector (middle), and “rescue transfected” (right). C Examples of di4 FLIM images of GPMVs generated from control, “scrambled” cells (left), EFR3A KnD cells transfected with an “empty” vector (middle), and EFR3A KnD cells transfected with a “rescue” plasmid p3xEFLAG-CMV10 EFR3A. D and E Quantitative distribution of fluorescence lifetime values obtained from FLIM analyses of di-4 labeled cells. D Plasma membrane and GPMVs. E FLIM analyses presented here were performed 48 h following transfection. F Dot-blot analysis of GPMVs derived from “rescue”-transfected EFR3A KnD cells using an anti-FLAG antibody. GPMVs were induced using a buffer containing NEM and CaCl2 48 h after transfection. Then, from the collected vesicles, proteins were precipitated with 10% TCA, washed, and resuspended in a reducing buffer containing DTT. Samples were applied on nitrocellulose using a dot blotter and probed with anti-FLAG antibodies