Fig. 5

Analysis of p-Smad2/3, Smad7 and IDE2 rescue assay in Rnf111-deficient embryos. (A, A’, A’’, B, B’, B’’) Double immunostaining of anti-GFP (A, B) and anti-p-Smad2/3 (A’, B’) in the caudal hematopoietic tissue of the transgenic Tg (cmyb-EGFP) line at 3 dpf. The bottom panel shows merged images (A’’, B’’). White arrows indicate p-Smad2/3 and cmyb-EGFP double-positive cells. (C) Statistics of p-Smad2/3 and cmyb-EGFP double-positive cells. The data are presented as mean ± standard deviation with ***P<0.001. (D, E) Immunohistochemistry of p-Smad2/3. (F) Western blot analysis of p-Smad2, Smad2, p-Smad3 and Smad3. (G) Western blot analysis of Smad7. (H) Whole-mount in situ hybridization analysis of rescue efficiency of IDE2. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells in the caudal hematopoietic tissue. All experiments were independently replicated at least three times. WT: wild-type; MO rnf111: rnf111 morphants; GFP: green fluorescent protein; MU: Rnf111 mutants with deletion of 4 base pair nucleotides; IDE2: definitive endoderm 2 inducer; EGFP: enhanced green fluorescent protein; dpf: days post-fertilization.