FIGURE 4:

Clathrin-mediated endocytosis is not a significant modulator of Lrp6 or Frizzled receptor abundance on the plasma membrane. (A) FACS analysis of mESCs stained with OMP-18R5 and detected with Alexa-488 secondary showed that CLTC knockdown does not alter cell surface level of Frizzled receptors. (B) Immunoprecipitation of biotinylated cell surface proteins was followed by quantitative Western blot to detect surface Lrp6 with or without 30-min stimulation with 5 nM Wnt3a. The cytoplasmic control GAPDH was detected in lysates but not in immunoprecipitated cell surface fractions. (C) Immunoprecipitation of biotinylated cell surface proteins was followed by quantitative Western blot to detect surface Lrp6 with or without 8-h stimulation with 5 nM Wnt3a. (D) Quantification of surface Lrp6 signal normalized by total Lrp6 signal in Western blots in B and C.