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Fig. S4 Validation and effects of two additional MOs targeting zebrafish TNFRSF1B. (A) RT-PCR analysis of TNFRSF1B-E1/I1-mo induced altered splicing of the tnfrsf1b transcript at 3 dpf. A dose-dependent decline of the wild type RNA was observed, suggesting the insertion of intron 1, which has 15,726 bp length. This insertion resulted in a predicted TNFRSF1B protein lacking both TNFα binding and signaling domains. Samples without template (-) gave no amplifications. The annealing of MO (dashed line) and the inframe premature stop codons (arrowheads) are indicated. (B) Embryos injected with 8 ng/egg (1 mM) TNFRSF1B-E1/I1-mo (middle panel) or TNFRSF1B-ATG-mo showed impaired differentiation of the caudal artery (CA) and caudal vein (CV) (asterisks), hemorrhages throughout the body (arrowheads) and altered blood circulation. A control larvae injected with STD-mo is shown on the left panel for comparison. Scale bars, 100 μm. (C) The vascular defects shown in (B) were scored at 72 hpf as indicated in the legend to Figure 2. ***P<0.0001 according to Chi-square contingency test.