Flower mutant cannot rescue the endocytosis defect. (A) Positions of the putative AP-2–binding motifs within Flower (red circles) with the WT and mutated sequences below. N, N terminus; C, C terminus. (B) Western blot (left panel, 4 to 20% SDS polyacrylamide gel) of surface-expressed Flower WT and mutant (mut) cDNAs in Cos-7 cells were detected by the Flower antibody. The ER protein calnexin was used as the control. One representative of six experiments that indicate more than threefold higher plasma membrane expression of the mutant protein compared with the WT protein (right; mean ± SEM; n = 6; **, P < 0.005, one sample t test). (C) SIM images (maximal-intensity projections [MIP] and single planes through the middle of the cell) of a CTL cotransfected with WT tagRFP-T-Flower (red) and Flower mutant-mTFP (green) constructs. Bar, 5 µm. (D) SIM images of SybKI CTLs (control) and Flower KO/SybKI CTLs in contact with target cells with endocytic anti-RFP647 (magenta). CTLs were incubated with target cells for 30 min in the presence of anti-RFP647 to visualize endocytic CGs. Flower KO/SybKI CTLs were transfected with Flower(WT)-mTFP and Flower(mut)-mTFP separately (bottom two rows) to observe the rescue. Bars, 5 µm. (E) Quantitative analysis from panel D of endocytic anti-RFP647 fluorescence signal at the IS. Data are given as mean ± SEM; unpaired Student’s t test: ***, P < 0.001.