Xenbase Image ID: 175396

Supplementary figure 2:Morpholino control experiments (A) Morpholino sequences of Cdk9a, Cdk9b and CyclinT1 shown to bind over the start site of their corresponding sequences. ATG start site is shown in red for all three morpholinos and sequences. (B) in vitro translation of Xenopus laevis CDK9.S, CDK9.L and CYCLINT1. Plasmid alone generates protein bands at expected sizes. Increasing amounts of morpholino 40ng, 80ng and 100ng was added to the CDK9.S, CDK9.L and CYCLINT1 in vitro translation showing a dose dependent decrease in the levels of protein detected. A luciferase protein was used as a positive control to ensure the in vitro translation is working. 100ng of each morpholino was added to this positive luciferase control to ensure the morpholinos will not knock down unspecific proteins. (C) To prove thatmorpholinos are specific rescue constructs were generated using site directed mutagenesis to mutate 4 bases around the ATG start site of the Cdk9.S, Cdk9.L and CyclinT1 plasmids. From these plasmids cRNA rescue constructs were generated which would not bind to the morpholinos. CDK9.S, CDK9.L and CYCLINT1 Morpholinos were injected alone or co-injected with 100pg of rescue constructs or 500pg of the rescue constructs. These embryos underwent Sox10 in situ as a read out of the rescue. Embryos were co- injected with 300pg of LacZ cRNA detected using red gal staining as a lineage tracer. All injections were carried out in one cell of a two cell stage embryo and in situ hybridisation was carried out at stage 16. (D) Graph showing the percentage of embryos displaying a wild type, partial loss or loss of Sox10 expression in the injected side after the rescue experiment. Cdk9.S mo n=67, cdk9.S mo 100pg rescue n=35, cdk9.S mo 500pg rescue n=50, cdk9.L mo n=45 cdk9.L mo 100pg rescue n=52, cdk9.L mo 500pg rescue n=23, cyclint1 mo n=59, cyclint1 mo 100pg rescue n=93, cyclint1 mo 500pg rescue n=31, como n=83. Image published in: Hatch VL et al. (2016) Copyright © 2016. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue sox10.L X. laevis Sometime during NF stage 15 to NF stage 16 neural plate border neural crest Image source: Published Larger Image Printer Friendly View

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