|ZFIN ID: ZDB-IMAGE-110526-8|
Fig. 2 Nkd1 co-localizes with Dvl2.
(A–F) Dvl2HA mRNA was injected at the one cell stage followed by injection of nkd1myc or nkd1G2A-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 Dvl2HA but negative for Nkd1myc (A–C) or Nkd1G2A-myc (D–F). Arrow in D–F indentifies a cell that is positive for both Dvl2HA and Nkd1G2A-myc. (G–H) Nkd1 interacts with Dvl2. (G) Embryos were injected at the one cell stage with nkd1myc or nkd1G2A-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) nkd1GFP or nkd1G2A-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 Nkd1myc 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.
|Acknowledgments:||ZFIN wishes to thank the journal for permission to reproduce figures from this article. Please note that this material may be protected by copyright.|