Fig. 3.

SHOX2 binds to regulatory elements within the ISL1 gene. A Schematic drawing showing the position of the luciferase reporter construct sequences within the genomic region of ISL1 and the probes generated for EMSA. Boxes and lines represent exons and introns, respectively. EMSA probes to which SHOX2 binds are indicated in red. B SHOX2 increases the transcriptional activity of the ISL1-reporter-2, as evaluated by luciferase assay. The responsive reporter element was further narrowed down using deletion (del) constructs. HEK 293 cells were transiently cotransfected with the indicated ISL1 reporter constructs or the empty pGL3 basic vector together with a SHOX2 expression plasmid or an empty vector (pST-Blue1) and 24 h after transfection, luciferase activity was determined. Experiments were repeated at least three times in triplicate with consistent results and representative data are shown. C EMSA revealed specific binding using Oligo1 and Oligo3 together with a GST-SHOX2 fusion protein (shifted band is marked by an arrowhead). Precise binding sites were identified by the use of mutated probes (Oligo1 mut and Oligo3 mut), with 4 nucleotides exchanged (AATT ↔ CCGG). Free oligo is indicated by an arrow. Representative data from three independent experiments with consistent results are shown