XB-IMG-148125
Xenbase Image ID: 148125
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Supplemental Figure 6. Characterization of GRAF-specific morphilinos. A,B) Flag-tagged human or
Xenopus GRAF cDNA constructs were subjected to an in vitro transcription/translation assay containing 35Smethionine
in the presence or absence of GRAF-specific anti-sense morpholinos (denoted mo1 and mo2) and
exposed to autoradiography (panel A, panel B top) or immunoblotted using GRAF-specific antibody (panel B,
bottom). Mo1 and mo2 each significantly reduced flag-tagged Xenopus GRAF1 expression in this in vitro
transcription/translation assay but had no effect on translation of a control plasmid encoding human GRAF1
(panel A). When injected individually, these morphilinos each led to lethality by stage 42 and all embryos
exhibited a similar range of phenotypes as observed when mixed together (i.e. fully penetrent motility defects,
cardiac edema, and a small percentage with shortened AP axis). Since, the mixture of the two morpholinos
(hereafter termed GRAF1 Mo) led to the most significant depletion of GRAF1 protein, (panel B) we used this
combination for the remaining studies limiting the potential for off-target effects. C. Whole-mount GRAF
antibody staining and laser scanning confocal microscopic analysis of Con Mo- (top) and GRAF Mo- (bottom)
injected embryos at stage 37 reveals that GRAF Ab recognizes GRAF protein by IHC. The specificity of this
staining is confirmed by markedly reduced immunoreactivity in GRAF morphant embryos. Dorsal is to the top,
anterior to the left Image published in: Doherty JT et al. (2011) Copyright © 2011. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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