Figure 3

Functional analysis of rs34623097. (a) Luciferase reporter assay of ADRB2 rs34623097 SNP. The reporter gene construct with rs34623097-A or rs34623097-G was transfected in HEK293T cells. Transcriptional activity was measured using Renilla luciferase as a reporter and was normalized using Firefly luciferase activity. The experiment was replicated five times, with quintuplicate assays performed within each replicate. The results of a total of 25 assays of each allele are divided by the mean of rs34623097-G. The values presented are mean±s.e. after the division (ie, relative values against rs34623097-G). The P-value was obtained by ANCOVA adjusted for replication. The significant difference in transcriptional activity between rs34623097-A and rs34623097-G was detected (P=7.5 × 10⁻⁶). (b) Electrophoretic mobility shift assay (EMSA) of ADRB2 rs34623097 SNP. The biotin-labeled probes carrying rs34623097-A and rs34623097-G were incubated with reaction mixtures containing no nuclear extracts (lanes 1 and 4) and with those containing 6 μg of nuclear extracts from HEK293T cells (lanes 2 and 5); lanes 3 and 6: as lanes 2 and 5 plus 200-fold molar excess of unlabeled probes carrying rs34623097-A and rs34623097-G.