Fig. 5.

Colocalization of WDFY2 with endocytic Rab GTPases. (A) HeLa cells were transiently transfected with RFP-Rab5Q79L (red), and after 24 h they were fixed and stained with antibodies (green) to EEA1 (Left) or WDFY2 (Right). Colocalization is depicted in yellow. (B) Cos-7 cells were fixed and stained with antibodies to WDFY2 or EEA1 (green) and antibodies (red) to Rab5 (Left), Rab4 (Center), and Rab11 (Right). Image stacks were obtained and restored; shown are single 250-nm-thick regions enriched in both signals. Colocalized regions are rendered in yellow. (C) The degree of colocalization of WDFY2 with each Rab, and of each Rab with WDFY2, was determined by using areas of the cell enriched in both signals, such as those shown in B. Colocalization is expressed as the percent of the total fluorescence from WDFY2 or EEA1 colocalizing with each Rab and vice versa. Spurious colocalization was obtained by flipping one of the images along the x axis. The value for spurious colocalization was subtracted from the properly aligned colocalization values. Bars represent the mean, and vertical lines indicate the SEM, from 12 regions (3 regions per cell) obtained from four independent cells. (Magnifications: A, ×2,000; B, ×3,000.)