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. Author manuscript; available in PMC: 2017 Dec 6.
Published in final edited form as: Hum Mutat. 2013 Jul 11;34(9):1226–1230. doi: 10.1002/humu.22366

Figure 2. Identification of FOXP1 c.1702C>T variation (p.Pro568Ser) in individual with CHD and functional analysis of murine Foxp1 P596S protein.

Figure 2

(A) Sequence chromatogram showing heterozygous C to T transition at nucleotide position 1702 in affected subject as compared to control individual. Asterisk (*) indicates the location of c.1702C>T transition as +1 corresponds to the A of the ATG of FOXP1 (GenBank accession number NM_032682.5). (B) Summary of non-synonymous FOXP1 p.Pro568Ser variation identified in children with CHD. (C) Luciferase reporter assays using the 3kb mouse Nkx2.5 promoter/enhancer demonstrates that the murine Foxp1 p.P596S mutant protein without wildtype Foxp1 does not repress the Nkx2.5-luciferase reporter as compared to wildtype Foxp1. The addition of Foxp1 p.P596S mutant did not affect the ability of wildtype Foxp1 to repress reporter activation. *, p<0.05 comparison between empty vector and Foxp1; #, p<0.05 comparison between wildtype Foxp1 and Foxp1 p.P596S. Luciferase activity is normalized to β-gal. (D) Expression of CyclinD1, CyclinD2, CyclinA2 and Nkx2.5 mRNA was increased with transfection of Foxp1 p.P596S plasmid alone as compared to wildtype Foxp1 in H9C2 cardiomyoblasts as measured by qRT-PCR. (E) Transient transfection of Foxp1 p.P596S mutation without wildtype Foxp1 is unable to repress proliferation of H9C2 cardiomyoblasts as compared to wildtype Foxp1 as measured by immunofluorescent staining for Ki67 and quantified in (F). DAPI is shown in blue. *, p<0.05. Scale bar indicates 100μm.