Fig. 2

Nkd1 co-localizes with Dvl2.

(A–F) Dvl2^HA mRNA was injected at the one cell stage followed by injection of nkd1^myc or nkd1^G2A-myc RNA into 1 of 4 blastomeres at the 4-cell stage. Embryos were harvested at 50% epiboly (5.25 hpf) and processed for immunohistochemistry using anti-myc probes (red) or anti-HA probes (green). Arrowheads in A-F identify a cell that is positive for Dvl2^HA but negative for Nkd1^myc (A–C) or Nkd1^G2A-myc (D–F). Arrow in D–F indentifies a cell that is positive for both Dvl2^HA and Nkd1^G2A-myc. (G–H) Nkd1 interacts with Dvl2. (G) Embryos were injected at the one cell stage with nkd1^myc or nkd1^G2A-myc and harvested at dome stage (4.3 hpf) for co-immunoprecipations. Lysates were incubated with anti-zDvl2 or anti-myc antibodies for immunoprecipitation. Immunoprecipitates were western blotted and probed with anti-zDvl2 or anti-myc antibodies (WB). Each lane represents the equivalent of 17.5 embryos. (H) nkd1^GFP or nkd1^G2A-GFP RNAs were co-injected with wnt8 RNA at the one cell stage. At 4.3 hpf, embryos were harvested and fractionated to isolate cytoplasmic (cyto) and plasma membrane (mem) fractions. Anti-actin and anti-pan-cadherin antibodies determined the purity of the cytoplasmic and plasma membrane fractions, respectively. A portion of the whole cell lysate was recovered prior to fractionation (lower panel) to determine loading controls. (Note: the high percentage gel precluded observing Dvl mobility shifts induced by Wnt8 in the whole cell lysate). The predicted MW of Nkd1^myc is 61 kD, but the observed MW is closer to 75 kD. We speculate that this is likely due to post-translational modifications of Nkd1. Each lane represents the equivalent of 1 embryo, averaged from 10 embryos. Scale bar represents 20 μm.