Fig. 4

Active Gata6 Can Compensate for Loss of Pak1

(A) Representative images of pak1 morphants that have been rescued by the addition of an active form of Gata6 (Gata6 SD) but not by the inactive mutant (Gata6 SA).

(B) Quantification of circulation in the Gata6 rescue. The indicated inducible Gata6 expression plasmids were coinjected with pak1 MO at the one-cell stage, followed by heat shock at 24 hpf as indicated.

(C) Fluorescent EMSA showing dose-dependent binding of WT Gata6 to a GATA-containing DNA template (EMSA) as well as effects of Gata6 phosphorylation on DNA binding.

(D) Quantitation of fluorescent EMSA showing that Gata6, when phosphorylated by ERK, is able to bind to target DNA more efficiently than unphosphorylated Gata6.

(E) Luciferase assay showing transcriptional effects of Gata6 using WT or mutant GATA reporters. p < 0.05.

(F) Luciferase assay comparing Gata6 SA and SD to WT. p < 0.05.

(G) Luciferase assays comparing active Pak1 (Pak L107F) and MEK DD, respectively, to control. p < 0.05.

(H) Luciferase assay comparing activated Pak1 plus either Gata6 WT or Gata6 SA. p < 0.05.

(I) WT embryos were injected with a vector encoding PID driven by the sox10 promoter plus an expression vector encoding either Gata6 SD or SA. Representative images were taken at 48 hpf.