XB-IMG-84556
Xenbase Image ID: 84556
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Fig. 3. PTK7 protein depletion inhibits LRP6 activity by reducing LRP6 protein levels. (A) Left panel: embryos were injected at the one-cell stage with PTK7-MO (10 ng) and RNA encoding the constitutively activated LRP6δN (170 pg) protein. ACs removed at the blastula stage were grown until gastrula st.10.5, when total RNA was isolated from six control embryos and 27 ACs from each group. Wnt direct-target gene expression was examined by sqRT-PCR. Right panel: embryos were injected at the one-cell stage with PTK7-MO (15 ng) and RNA encoding the full-length VSVG tagged LRP6 (2 ng) protein. ACs removed at the blastula stage were grown to gastrula st.10.5, when total RNA was isolated from six control and injected embryos and 27 AC explants from each group. Wnt direct-target gene expression was examined by sqRT-PCR. (B) Embryos were injected at the one-cell stage with RNA encoding the Wnt3a (5 pg, lanes 8-10) or LRP6δN (100 pg, lanes 11-13) proteins. These embryos were than co-injected with either the PTK7-MO (10 ng, lanes 6,9,12) or RNA encoding the dominant-negative Dvl protein (Xdd1, 0.7 ng, lanes 7,10,13). ACs removed at the blastula stage were grown to gastrula st.10.5, when total RNA was isolated from six control and injected embryos (lanes 2-4) and 18 ACs from each group. Wnt direct-target gene expression was examined by sqRT-PCR. (C) Embryos were injected at the one-cell stage with PTK7-MO (13 ng) and RNA encoding the constitutively activated LRP6δN (200 and 300 pg) protein. ACs removed at the blastula stage were grown to gastrula st.10.5. Total protein was isolated from 27 ACs per group for western analysis. The T1479 antibody recognizes unphosphorylated and phosphorylated forms of LRP6δN. (D) LRP6 induction of Wnt activity is PTK7 dependent. HEK293 cells were transfected with the Topflash-luc reporter, which was activated by co-transfection of LRP6 encoding DNA (0.2 μg). Co-transfection of a neutral Renilla vector serves as an internal control. A PTK7-targeting shRNA used to deplete endogenous PTK7 protein was compared with a nontargeting control shRNA. Increasing amounts of Xenopus PTK7 encoding DNA were transfected as indicated. Data are presented as fold activation; the signal induced by LRP6 is presented as 1. Fold-activation from seven independent experiments is shown. Image published in: Bin-Nun N et al. (2014) Copyright © 2014. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |