Fig. 6. O-fut1 is secreted and internalized by cells. (A,B) Intracellular localization of O-fut1 was examined in Garland cells overexpressing either ER-CFP (A, green) or N⁺-GV3 (B, green) and Ofut1-myc (A,B, magenta). ER-CFP and O-fut1-myc were driven by da-GAL4, and N⁺-GV3 was induced by a 2 hour heat shock that ended 30 minutes before fixation. (A',B') Higher magnifications of A and B, respectively. Arrows indicate O-fut1 and Notch double-positive vesicles, which were distant from the peri-nuclear ER (B'). (C) S2 cells were transfected with the expression constructs for O-fut1-myc and Notch (lanes 1,2), or Notch^EC (lanes 3,4). Lysates prepared from these cells were immunoprecipitated using anti-Myc (lanes 1,3) or anti-Notch (lanes 2,4) antibodies, and the blots were probed with the anti-Notch antibody (C17.9C6) or anti-Myc antibody (9E10). The expression of Notch and O-fut1 in the lysates was confirmed by western blot (Lysate, lower panels). (D) O-fut1 was incorporated into cells in a Notch-dependent manner. S2 cells expressing Notch (green) were incubated with the O-fut1-ER^- protein (magenta). The brightfield image is superimposed in the right panel. (E-H) The endocytosis defect in the Ofut1 knockdown cells was rescued by the addition of extracellular Ofut1. S2 cells expressing Notch (E) or Notch and O-fut1-IR (F,G) were incubated with anti-Notch antibody (rat1, magenta), fixed, and costained with anti-Hrs antibody (green). (G) Live cells expressing Notch and O-fut1-IR were incubated with O-fut1-ER^- for 20 minutes before the addition of anti-Notch antibody. (E'-G') Corresponding higher magnification images of E-G. Arrows indicate Notch and Hrs double-positive vesicles. (H) Average ratio of Notch and Hrs double-positive vesicles shown as the percentage of Notch-positive vesicles. Mean ± s.d. from triplicate assays (more than 20 cells each) are shown. (I,J) The N⁺-GV3 protein (magenta) 8 hours after heat shock for 1 hour in thirdinstar wing discs overexpressing O-fut1 (I) or O-fut1-ER^- (J) (green) driven by en-GAL4. (K,L) Adult wings. O-fut1 (K) and O-fut1-ER^- (L) were overexpressed under the control of en-GAL4, and the resulting wing phenotypes were examined. (M) Fluorescent dextran uptake by Delta and Serrate double-mutant cells in live wing discs after a 10 minute incubation and a 20 minute chase at 25°C. Clone boundary and dextran-positive vesicles are indicated by dotted lines and white arrowheads, respectively. All wing discs were isolated from late thirdinstar larvae.