Fig. S3

Transient knockdown of nf1a and nf1b is achieved by specific translational- and splice-blocking MOs. (A) Western blot analysis of nf1a ATG 5MP MO (2 ng)-, nf1a ATG MO (2 ng)-, nf1b ATG 5MP MO (10 ng)-, nf1b ATG MO (10 ng)-, nf1a + nf1b ATG 5MP MO (2 ng)-, and nf1a + nf1b ATG MO (2 ng)-treated 3.5-dpf zebrafish embryos. Administration of MOs specific for nf1a, nf1b, or both together leads to a marked decrease in neurofibromin at the protein level (wild-type and Nf1-/- mouse lysates are included as controls for antibody specificity). (B) Activation of effector pathways downstream of Ras, as assessed by increased P-p44/42 MAPK, are observed by Western blot analysis in 3.5-dpf nf1a (≈3 ng), nf1b (≈5 ng), and nf1a+nf1b (≈2 ng) ATG morphant zebrafish embryos when compared with dose-matched controls. (C) Schematic representation of site targeted by nf1a-SBe1MO.Administration of nf1a-SBe1MOleads to activation of a cryptic splice donor and the generation of an mRNA transcript harboring a premature stop codon. MO-mediated inclusion of intronic sequence in the mRNA transcript generates a 673-bp PCR product by RT-PCR using the depicted primer pair. (D) RT-PCR analysis from 1-, 2-, 3-, and 5-ng nf1a-SBe1 MO-treated samples using the depicted primer pair reveals a dose-dependent increase in the amount of the 673-bp PCR product that is absent in uninjected embryos. Injection of nf1a-SBe1 5MP MO does not lead to the generation of the 673-bp PCR product. (E) Quantitative PCR analysis of uninjected, nf1a-SBe1 MO-, and nf1a-SBe1 5MP MO-treated samples using the depicted primer pair (mean fold change ± SD). Administration of 3 ng of an nf1a-SBe1 MO leads to a 77% reduction in the wild-type nf1a transcript when compared with uninjected or 3-ng-injected nf1a-SBe1 5MP MO-treated samples. (F) Schematic representation of site targeted by an nf1b-SBe4 MO. Administration of nf1b-SBe4 MO leads to the inclusion of intron 4/5 in the nf1b mRNA transcript, which harbors a premature stop codon. MO-mediated inclusion of intronic sequence in the mRNA transcript generates a 1,497-bp PCR product by RT-PCR using the depicted primer pair, whereas the wild-type transcript generates a 63-bp PCR product. (G) Quantitative PCR analysis of uninjected, nf1b-SBe4 MO-, and nf1b-SBe4 5MP MO-treated samples using the depicted primer pair (mean fold change ± SD). Administration of 1, 2, or 4 ng of nf1b-SBe4 MO leads to a dose-dependent decrease in the wild-type nf1b mRNA transcript when compared with uninjected or nf1b-SBe4 5MP MO-treated samples.