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Fig. 8 | BMC Evolutionary Biology

Fig. 8

From: Lineage-specific duplication of amphioxus retinoic acid degrading enzymes (CYP26) resulted in sub-functionalization of patterning and homeostatic roles

Fig. 8

In vitro validation of putative retinoic acid response elements (RAREs) in the amphioxus CYP26 cluster. Using in vitro synthesized Branchiostoma lanceolatum RAR and RXR proteins, the binding of the cognate RAR/RXR heterodimer to putative RAREs identified in the amphioxus CYP26 cluster was assessed. The mouse CYP26A1 DR5 sequence (R1) [26] (Table 1) was used as 32P-radiolabeled double-stranded probe. The RXR and RAR lanes respectively contained only in vitro synthesized RXR or RAR proteins, together with the radiolabeled probe. The H20 (water) lane contained radiolabeled probe as well as RAR and RXR proteins. The NS (non-specific) lane contained a non-specific and unlabeled double-stranded DR element in 100-fold molar excess relative to the radiolabeled probe as well as RAR and RXR protein. All other lanes contained one of the 16 putative amphioxus DR elements (for nomenclature explanations see Fig. 7b) at 10-fold (left lane) or 100-fold (right lane) molar excess, along with RAR and RXR protein and the radiolabeled mouse R1 element, to test the binding specificity of the putative amphioxus RAREs. A black asterisk indicates that a given DR element can be recognized by the B. lanceolatum RAR/RXR heterodimer, as it outcompetes binding to the mouse R1 element

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