FIGURE 2.

Surface-labeled Notch1 is constitutively endocytosed into vesicles that colocalize with FITC-dextran and HRS. A, C2C12 cells that stably express HA-tagged Notch1 were surface-labeled with Al488anti-HA at 4 °C (left panel), washed, and incubated at 37 °C for 60 min to allow for endocytosis (right panel). B, HEK293T cells transiently expressing HA-Notch1 were incubated for 18 h with 1 mg/ml FITC-dextran (70,000 molecular weight; Molecular Probes) in normal growth media at 37 °C. The cells were washed and chased with media containing anti-HA for 2 h, fixed, permeabilized, and then stained with Cy3-α-mouse to identify α-HA-labeled Notch1 and with rabbit α-NbC (Cy5-α-rabbit secondary) to mark the plasma membrane. C, HEK293T cells transiently expressing HA-Notch1 were incubated with Alexa488-α-HA (green) at 37 °C for 1 h. Following fixation with 2% paraformaldehyde, cells were permeabilized and immunostained with rabbit αHRS (Cy5-labeled donkey α-rabbit) and guinea pig αNbC (Cy3 labeled donkey anti-guinea pig). Images were captured using a Zeiss Axiovert 200 microscope equipped with a Hamamatsu Orca AG CCD camera and spinning disk confocal scan head. Images were processed using Volocity software, and figures were prepared using Photoshop. Scale bars represent 10 μm. Images are representative of three independent experiments.