Fig. 5

PGE₂/Gβγ signaling stabilizes Snai1 by inhibiting Gsk3β-mediated proteasomal degradation. (A-E) Confocal images of zebrafish embryos in which the Snai1a-YFP assay was used to test downstream components of PGE₂ signaling for their ability to affect levels of Snai1a in control (top row) and ptges morphants (middle and bottom rows). (A) Treatment with the proteasome inhibitor MG132. (B) Injection of zebrafish gsk3β RNA and treatment with the Gsk3β inhibitors LiCl and BIO. (C) Injection of human Gβ₁ and Gγ₂ RNA. (D) Inhibition of both Gsk3β and the proteasome (MG132 treatment). (E) human Gβ₁γ₂ and zebrafish gsk3β RNA co-injection. (F) Snai1a-YFP expression in control (top row) and ptges morphants (bottom row) injected with RNA encoding constitutively activeΔNβ-catenin. (G) Immunoblotting of Gsk3β following immunoprecipitation with human Gβ₁γ₂. Zebrafish Gsk3β and HA-tagged human Gβ₁γ₂ were transfected into HEK-293T cells. Cell extracts were immunoprecipitated with anti-HA, then immunoblotted with anti-Gsk3β (Gsk3β indicated by arrowhead). The interaction between Gsk3β and human Gβ₁γ₂ was blocked by the addition of c-βark (top). Total lysate western blots are shown at the bottom. (H) Immunoblotting of Gsk3β following PGE₂ treatment and immunoprecipitation with human Gβ₁γ₂. Zebrafish Gsk3β and HA-tagged human Gβ₁γ₂ were transfected into HEK-293T cells, then cells were treated with 0.1, 1 and 10 μM PGE₂ prior to collecting the cell extracts in NDLB. Cell extracts were immunoprecipitated with anti-HA, then immunoblotted with anti-Gsk3β. Total lysate western blots are shown at the bottom.