Fig. 3. PTK7 is required for fz7-mediated dsh recruitment and phosphorylation. (A-D) PTK7 is required for fz7-mediated dsh recruitment to the plasma membrane. GFP-tagged dsh is shown in green, myc-tagged fz7 in red, HA-tagged PTK7 in blue. (A) GFP-tagged dsh is localized to the plasma membrane in animal caps injected with 100 pg dsh-GFP RNA, 100 pg fz7-myc RNA and 20 ng control MO. (B) GFP-tagged dsh is not recruited to the plasma membrane in animal caps injected with 100 pg dsh-GFP and 100 pg fz7-myc RNA and 20 ng PTK7 MO. (C) Co-injection of 100 pg wild-type PTK7 RNA lacking the MO binding site rescues dsh-localization of animal caps injected with 100 pg dsh-GFP RNA, 100 pg fz7-myc RNA and 20 ng PTK7 MO. (D) Graph summarizing the percentage of cells with simultaneously membrane-localized dsh and fz7. The total number of cells is indicated on each column. (E) PTK7 is required for fz7-dependent hyperphosphorylation of dsh. Embryos were injected with 100 pg dsh-myc RNA, 100 pg fz7 RNA, 500 pg PTK7-myc RNA, 20 ng control MO or 20 ng PTK7 MO in the combinations indicated. Animal caps were cut at stage 9 and their lysates were analyzed by western blotting using anti-myc antibodies. Hyperphosphorylated dsh is detected as a second high molecular weight band. One representative experiment of three independent experiments is shown.