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Figure S4. Xenopus laevis morpholino oligonucleotides (MO) and vivo-morpholino oligonucleotides (vivoMO) efficiency and specificity tests.
(A) Scheme of experiments on testing MO/vivoMO. mRNA encoding for Flag-tagged xRas-dva 1 or 2 was injected into each blastomere of 2-cell Xenopus laevis embryos (100 pg/blastomere), either alone or with control mis-xRas-dva1 vivoMO (mis-xRAs-dva2 vivoMO) or with specific xRas-dva1 MO/vivoMO (xRas-dva2 MO/vivoMO) (4nl of 0,25mM MO solution or 4 nl of 0,4 mM water vivoMO solution per blastomere). The injected embryos were collected at the late gastrula stage and analyzed for presence of 3Flag-xRas-dva proteins by Western blotting with
anti-Flag antibody. Tubulin was used as loading control (see Methods). Drawings were done by M.B.T. (B) Results of western blotting with conjugated anti-flag alkaline phosphatase antibody (Sigma) and monoclonal anti-tubulin antibody demonstrate specific and effective inhibition of flag-xRas-dva1 synthesis by xRas-dva1 MO, but not by xRas-dva2 MO. The common results are obtained for xRas-dva2 MO demonstrating their effectiveness and specificity. (C) Results of western blotting with conjugated anti-flag alkaline phosphatase antibody (Sigma) and monoclonal anti-tubulin antibody demonstrate specific and effective inhibition of flag-xRas-dva1 synthesis by xRas-dva1 vivoMO, but not by xRas-dva2 vivoMO or mis-xRas-dva1 vivoMO. The common results are obtained for xRas-dva2 vivoMO. Image published in: Ivanova AS et al. (2018) © The Author(s) 2018. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |