Figure 3

Wnt signaling is dependent upon clathrin-mediated endocytosis. (a) L cells were stimulated for 3 hours in the presence or absence of Wnt-3A (~100 ng/mL), control conditioned medium (Cont. CM), or Wg conditioned medium (Wg CM) and subsequently harvested and assayed for β-catenin and GSK3β levels by immunoblotting. β-catenin and GSK3β levels were similarly assessed in L cells stimulated with Wnt-3A over a 2-hour time-course in the presence or absence of MDC (300 μM) (b), hypertonic sucrose (0.45 M) (c), or CPZ (30 μM) (d). (e) LSL cells were stimulated for 5 hours in the presence or absence of Wnt3A with either DMSO (Vehicle), MDC, hypertonic sucrose, or CPZ and assayed for TOPFLASH reporter activity. Shown is a representative experiment from 3 independent determinations each performed in triplicate, in which values represent means ± SEM. (f) L cells were pre-incubated with or without DMSO Vehicle (Veh), MDC, hypertonic sucrose, or CPZ for 1 hour, followed by stimulation in the presence or absence of LiCl (50 mM) for 3 hours. Cells were subsequently harvested and assayed for β-catenin and GSK3β levels by immunoblotting. (g) SW480 cells were treated for 1 hour with Vehicle, MDC, hypertonic sucrose, or CPZ and were subsequently lysed under hypotonic conditions to enable measurement of cytosolic β-catenin and GSK3β levels.