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. a, Electrophoretic mobility shift assay 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 electrophoretic mobility shift assay. 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 and 4) render the probe unable to bind nuclear extracts from zebrafish brain, whereas 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).