Fig. s2

Myc-tagged zebrafish RARs are expressed in zebrafish embryos. (A) Western blot of myc-tagged Hs dnRAR and zebrafish WT RARs and dnRARs at 70–90% epiboly in zebrafish embryos that were injected with the indicated mRNAs. The decreasing relative expression from left to right reflects the timing of execution for this representative experiment, in which constructs on the left were injected earlier than those on the right. Approximate sizes of the myc-tagged proteins are: Hs myc-dnRARa, 55 kDa; Dr myc-RARab, 61 kDa; Dr myc-dnRARab, 55 kDa; Dr myc-RARga, 67 kDa; Dr myc-dnRARga, 52 kDa. The approximate size of α-tubulin is 50 kDa. (B–D) Wholemount immunofluorescence at 70–90% epiboly of zebrafish embryos that were injected with mRNAs for myc-tagged human and zebrafish RARs. Exogenous protein expression was analyzed at mid- to late-gastrulation stages because this is when RA signaling is required to pattern the early zebrafish embryo ([Hernandez et al., 2007], [Hernandez et al., 2004], [Linville et al., 2009], [Maves and Kimmel, 2005] and [Stafford et al., 2006]).Both analyses indicate that, while there may be differences in precise expression levels of different exogenous RARs, all of the exogenous RARs are expressed broadly and robustly in zebrafish embryos at these stages. Unfortunately, there are no commercially available anti-RAR antibodies that allow evaluation of the endogenous levels of zebrafish RARs. If we presume that endogenous α-tubulin is likely to be present at higher levels than endogenous RARs, the qualitative comparison of exogenous RARs and endogenous α-tubulin suggests that the exogenous RARs are present in excess of their endogenous counterparts.