IMAGE

Fig. S3

ID
ZDB-IMAGE-101116-2
Source
Figures for Padmanabhan et al., 2009
Image
Figure Caption

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Proc. Natl. Acad. Sci. USA