Fig. 4. Quantification of the de novo formation of caveolae in mammalian cells. (A-F) Cav1-null MEFs transiently transfected with CfCav1-YFP (A,F), GFP (B,F), HsCav1β-GFP (C,F) CeCav-a-GFP (D,F), HCav^49-132-GFP (F) or co-transfected with flotillin1-GFP and flotillin2-RFP (E,F), were FAC sorted by fluorescent intensity into two pools with different expression levels with the exception of flotillin-1 and flotillin-2. The cells were fixed and examined by electron microscopy. Caveolae (arrows) were only observed in cells transfected with CfCav1-GFP, HsCav1β-GFP and at a lower frequency in cells expressing HCav^49-132-GFP. Clathrin-coated pits are highlighted by arrowheads. Scale bars: 200 nm. (F) Caveolae-like structures as determined by morphology were counted at the plasma membrane and the density of caveolae-like structures was determined. ^**P0.05, difference between high and low expression pools of the same construct.