Abi-dependent CME is required for ligand-induced Notch internalization and signaling activation. (A) Single confocal slices of primary hemocytes from HmlΔ-GAL4,UAS-EGFP/+ (WT), UAS-abi^RNAi/+; HmlΔ-GAL4,UAS-EGFP/+ (HmlΔ > abi^RNAi), HmlΔ-GAL4,UAS-EGFP/UAS-Chc^RNAi (HmlΔ > Chc^RNAi), HmlΔ-GAL4,UAS-EGFP/UAS-Rabankyrin^RNAi (HmlΔ > Rabankyrin^RNAi) late-third instar larvae. Primary hemocytes were cocultured with S2S cells, and subjected to a Notch internalization assay as in Fig. 4 D. Arrowheads indicate intracellular punctate structures containing internalized Notch. (B) Quantification of the ratio of internal to surface Notch fluorescence intensities. n = 9 hemocytes. (C) Single confocal slices of S2N cells transfected with NRE-GFP with or without abi, Chc, or Rabankyrin dsRNA and cocultured with S2S cells for 6 h, prior to immunofluorescence analysis using anti-GFP (green), anti-NECD (pseudocolored magenta), and anti-Myc (for Myc-Ser on S2S cells; blue) antibodies and DAPI (white). (D) Quantification of the ratio of mean GFP to DAPI fluorescence intensities. Values are percentages of mock-transfected isolated (-Ser) cells. n = 9 cells. (E) Bright-field images of heated (70°C, 10 min) HmlΔ-GAL4/+, HmlΔ-GAL4/UAS-Chc^RNAi (HmlΔ > Chc^RNAi), and HmlΔ-GAL4/UAS-Rabankyrin^RNAi (HmlΔ > Rabankyrin^RNAi) late-third instar larvae. Dorsal views of the two most posterior segments (A7 and A8). (F) Numbers of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. Data represent the mean ± SEM. Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test (***P < 0.001). Scale bars: 10 μm (A and C); 200 μm (E).