Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease
J. Clin. Invest. Hideko Kasahara, et al. 106:299 doi:10.1172/JCI9860 [Go to this article.]

Figure 2
Expression of translated products in cells: intron-splicing defect in M112 mutant resulted in poor protein accumulation. (a) Wild-type and mutant CSX/NKX2.5 expression vectors were transfected into COS 7 cells, and the protein expression was examined by Western blotting using anti-FLAG Ab approximately 24 hours after transfection (FLAG, top). All mutant proteins except M112 (lane 11, asterisk indicates the expected molecular weight of M112 protein) were detected at the expected molecular weight. GAPDH expression in each lane was also shown (GAPDH, bottom). (b) Wild-type and mutant CSX/NKX2.5 proteins accumulated in the nucleus colocalizing with Hoechst nuclear staining (NUC, lower panels). The results presented are wild-type, group 1 (M170), group 2 (M191), group 3 (M198), and group 4 (M25). (c) G→T substitution (large arrow) identified in M112 mutant on the CSX/NKX2.5 splicing donor site was examined by RT-PCR. RNA-purified form COS 7 cells transfected with the wild-type and M112 mutant of CSX/NKX2.5 were amplified with two primers spanning the intron. In the wild-type transfectant, the intron was spliced out, resulting in the generation of 240-bp product, whereas in the mutant transfectant, G→T substitution of the first codon of the intron splicing site abolished the normal splicing, resulting in generation of 1,779-bp product. CSX/NKX2.5 protein was encoded by the two exons represented. (d) Translated products were examined approximately 24 hours after transfection by Western blotting using anti-FLAG mAb (top) and anti-Csx/Nkx2.5 mAb (bottom). Wild-type CSX/NKX2.5 gene was translated into approximately 42-kDa protein and was detected with anti-FLAG and anti-Csx/Nkx2.5 mAb (lane 1). In contrast, M112 mutant protein that is expected to migrate approximately 29 kDa was not detected in the cell lysate (lane 2). In in vitro transcription and translation system, cDNA produced approximately 42-kDa protein (lane 1), and M112 genomic construct produced about 29-kDa protein (lane 2).