Fig. 4.

The interaction between Vangl2 and Prickle3 is enhanced in Fz3-depleted embryos. (A) Experimental scheme. 4- to 8-cell embryos were sequentially injected into dorsal animal blastomeres with BLN–Pk3 and HA–Vangl2 DNAs (50 pg each), and Fz3 MO1 or Fz3 MO2 with biotin. Embryo lysates were collected at stage 18 for immunoprecipitation (IP) and immunoblotting (IB). (B) Vangl2 and Pk3 biotinylation in control and Fz3 morphant embryos in pulldowns were assessed with anti-biotin antibodies. Total exogenous Vangl2 levels were analyzed by anti-HA antibodies. Owing to low abundance and insufficient sensitivity of the detection, the levels and the activity of FLAG–BLN–Pk3 were assessed in the second sequential FLAG pulldown. Biological triplicates were performed for each experimental condition with 20 embryos per sample. (C) Band intensity ratios of biotinylated to total Vangl2 levels in pulldowns, evaluated with anti-biotin and anti-HA antibodies, respectively. Results are mean±s.d. (n=3). *P<0.05; **P<0.001 (two-tailed unpaired Student's t-test). (D) Anti-Fz3 antibody detects Fz3 (arrowhead) in lysates of stage 16 control embryos or embryos injected with Fz3-FLAG mRNA (25 pg); asterisks indicate non-specific bands. (E) Levels of the endogenous Fz3 protein (arrowhead) in control, Fz3 MO1- and Fz3 MO2-injected stage 16 embryos; ERK1 is a loading control. Data in D,E are representative of three experiments.