FIGURE 8.

Unbiased quantitative analysis of LGR5 internalization reveals the C-terminal motif responsible for internalization. HEK cells were transfected with the constructs utilized according to Fig. 1 (A), Fig. 4 (B), Fig. 5 (C), Fig. 6 (D), and Fig. 7 (E). Cells were pulse-chased at 37 °C with primary MSαHA antibody fixed and then stained with a GαM680 without permeabilization to assess the fraction of the receptor pulsed that remained on the surface following the chase. Cells were chased for (A) 0, 3.75, 7.5, 15, 30, or 120 min or (B–E) 0, 7.5, 15, 30, or 120 min. Cells were imaged on a LiCOR Odyssey and data normalized to the receptor on the cell surface at time 0 for each construct. F, data from each receptor construct were log2-transformed and normalized to the geometric average of the FL-LGR5 construct and presented as a heat map over the internalization time course (0, 7.5, 15, 30, and 120 min) where bright magenta indicates 100% cell surface expression and bright yellow indicates 8.4% cell surface expression. Reference values for cell surface expression and their correlation to color are indicated on the map.