Fig. 7

Ajuba Regulates Nkx2.5 Levels(A) In situ hybridization for Nkx2.5 in control and Ajuba morphant embryos at 10 and 15 somites.(B) Relative expression level of Nkx2.5 normalized to Rpl13α in pools of control and Ajuba morphant embryos at 10 and 15 somites. Error bars represent SDs derived from three independent pools of control (n = 10) and Ajuba morphant embryos (n = 10). **p < 0.01.(C) FLAG-HA-tagged Ajuba and Nkx2.5-myc expression plasmids were transiently expressed in HEK293T cells, and immunoprecipitation with an anti-HA antibody followed by immunoblot analysis with an anti-myc antibody was performed.(D) Western blot analysis of lysates from day 5 EBs overexpressing either GFP only or both GFP and Ajuba using Nkx2.5 antibody. The two lanes were spliced from the same exposure of the same blot.(E) HEK293T cells were transfected with constant amounts of Nkx2.5-myc (5 μg) and FLAG-H3.3 (1 μg, used as a control for transfection efficiency) expression plasmids and increasing amounts of an Ajuba plasmid (0.25, 0.5, 0.75 μg). Immunoblot analysis of equal amounts of total protein extracts was performed using either anti-myc, anti-HA or anti-FLAG antibodies.(F) Western blot analysis of whole-cell lysates from HEK293T cells treated with either DMSO or MG-132 using Nkx2.5 antibody. Ponceau staining served as loading control.(G) In situ hybridization of control and Nkx2.5/Nkx2.7 morphant (Nkx-deficient) embryos at 3, 5, 10, and 15 somite stages and at 48 hpf with an Isl1 probe.(H) Model for the control of the SHF progenitor pool by Ajuba.