Figure 7.

The TBX5/HAND2 Functional Interaction Inhibited both by the p.G202V variant and Thalidomide. The transcriptional activity of HAND2 was assessed in co-transfection assays using the rat NPPA promoter coupled to luciferase which showed a drastic decrease in the p.G202V mutant ability to activate its target (A). Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0.05) was assessed using the one‐way ANOVA test. HA‐tagged TBX5 and Flag‐tagged HAND2 (WT and G202V) proteins were extracted from transiently transfected HEK293 cells. Nuclear lysates for both HAND2 and TBX5 proteins were immune-precipitated with anti‐HA antibody, and visualized with western blot via anti‐flag antibody. Membrane stripping and subsequent western blot analysis was performed with anti‐HA antibody in order to detect TBX5 proteins. Ten times the quantity of proteins loaded for western blot was used for immune-precipitation. Quantitation of the bands shows up to 80% inhibition of the interaction when the HAND2 mutated protein is used instead of the wild type protein (B). Transcriptional activity was assessed by luciferase assay (C,D). TBX5 and HAND2 (WT or G202V) were transiently co-transfected with the rat NPPA promoter coupled to luciferase in HEK293 cells (C), then in the presence of Thalidomide (D). Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0.05) was assessed using the one‐way ANOVA test. (+) is for 300 ng of DNA, and (++) for 700 ng of DNA. The increasing doses start at 100 reaching up to 500 ng.