Haploinsufficiency of Rps14 or Rps19 in zebrafish result in phosphorylation of S6K1. (A) Zebrafish embryos were injected with rps14, rps19 and control MOs. Twenty-four hpf, embryos were treated with egg water (−) or with 100mM L-leucine (+) and incubated for an additional 24 hours. Embryos were then manually deyolked and the total protein isolated, subjected to Western blot analysis and probed sequentially with antibodies for p-S6K1^T389, p-4E-BP1^T37/46, and β-actin. (B-G) Whole mount staining of zebrafish embryos with p-S6 antibody. In control morphants L-leucine treatment results in a modest increase in pS6 which is apparent throughout the embryo. In rps14 (E) and rps19 (G) morphants, clusters of presumed hematopoietic progenitor cells expressing high levels of p-S6 are present in the caudal hematopoietic tissue (E-G red arrows) Number in brackets in the number of embryos showing the phenotype in the image. (H) Embryos were injected with control or rps19 MOs and treated with 1mM rapamycin or 1mM rapamycin + 100mM L-leucine at 24 hpf. Western blot analysis was performed 24 hours later. A DMSO control (diluent for rapamycin) was also included. The blot was sequentially probed with antibodies for p-S6K1^T389, S6K1, and β-actin. (I) Embryos were injected with control or rps14 MO and treated with rapamycin and rapamycin+L-leucine as in panel B. The blot was sequentially probed with the same antibodies as in panel B.