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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: J Neurosci. 2008 Jan 30;28(5):1109–1117. doi: 10.1523/JNEUROSCI.4853-07.2008

Figure 2. An E-box within the 109 bp region that is required for transgene expression in Müller glia binds nuclear extracts from zebrafish and rat brain and zebrafish retina.

Figure 2

(a) Electrophoretic mobility shift assay (EMSA) using a probe from the 109 bp region binds nuclear extracts from zebrafish and rat brain. The arrow indicates specific binding. Cold indicates where 50-fold molar excess unlabeled probe was added as competition. Extract indicates whether zebrafish (zf) or rat brain extracts were added. (b) Nucleotide sequence of the probes used for EMSA. The E-box is outlined in probe 4 with a box. Mutations are indicated by italicized and underlined text. (c) Mutations to the E-box (lanes 3 & 4) render the probe unable to bind nuclear extracts from zebrafish brain, while mutations to non-E-box nucleotides do not affect binding (lanes 1, 2 and 5). Probes correspond to those shown in (b). Unlabeled mutant probes compete with wild-type probe binding when the E-box is intact (lanes 6, 7 and 10), but not when the E-box is mutated (lanes 8 and 9), even at 50-fold molar excess. (d) Nuclear extracts from zebrafish retina bind specifically to the E-box. (e) An E-box probe from a different region of the promoter (Eb) is unable to compete with the E-box from probe 4 (lane 3), and does not bind to zebrafish brain nuclear extracts (lane 4).