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. 2023 Jun 20;299(8):104945. doi: 10.1016/j.jbc.2023.104945

Figure 5.

Figure 5

Fluorescence protease protection confirms cytosolic positioning of N and C terminal tails of hFWE3 and hFWE4. HEK293 stably expressing EGFP, EGFP-hFWE3, hFWE3-EGFP, EGFP-hFWE4, hFWE4-EGFP, or transiently transfected with 3xHA-LGR5-EGFP or mCherry-ER-3 were incubated in the presence or absence of 80 μg/ml digitonin for 90 s before removal and incubation in KHM buffer with or without 50 μg/ml proteinase K. 20× live confocal imaging at 1 s intervals for native EGFP or mCherry was initiated upon addition of digitonin and continued out to 400 s. Confocal images presented are representative of t = 0, 90, and 280 s time points for each experimental group, scale bar = 20 μM (N ≥ 3 independent timelapses per construct). Red arrowheads indicate representative cells across the three timepoints.