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Fig. S4 Rack1 is associated with Vangl2 in zebrafish embryo extracts. RNA encoding EGFP-Vangl2 (200 pg) was injected into zebrafish embryos at the one- or two-cell stage. The embryos were collected from the bud stage (10 hpf) to 10-somite stage (14 hpf) and processed for yolk removal and protein extraction from the dissected EGFP-Vangl2–expressing embryos. Similarly, extracts were made from control embryos. (A) Coimmunoprecipitation experiments were performed as described. Note that EGFP-Vangl2 was pulled down only in the presence of Rack1 antibody and that Rack1 was coimmunoprecipitated only in the presence of EGFP-Vangl2 and EGFP antibody. (B) The protein extracts from the control and EGFP-Vangl2–injected embryos were loaded directly and blotted with an antibody against Rack1 as input controls.