Figure 1.
Functional analysis of IKAROS C126W mutation. (Left) Immunofluorescence analysis of pericentromeric heterochromatin localization (PC-HC) demonstrating nuclear distribution of wild-type (WT) and C126W IKAROS. WT IKAROS shows the characteristic punctate nuclear pattern, whereas the C126W variant displays diffuse nuclear staining, indicating impaired DNA binding and altered pericentromeric localization with no evidence of a dominant-negative effect on the WT/Mutant co-transfection experiment. (Right) Electrophoretic mobility-shift assay (EMSA) using IKBS1, IKBS4, and gSat8 probes demonstrates that wild-type IKAROS binds consensus DNA sequences, whereas the C126W variant fails to bind or is markedly reduced. Together, these findings confirm that the C126W mutation disrupts IKAROS pericentromeric heterochromatin localization and DNA-binding capacity, consistent with a loss-of-function, haploinsufficiency mechanism. Refer to the image caption for details.

Functional analysis of IKAROS C126W mutation. (Left) Immunofluorescence analysis of pericentromeric heterochromatin localization (PC-HC) demonstrating nuclear distribution of wild-type (WT) and C126W IKAROS. WT IKAROS shows the characteristic punctate nuclear pattern, whereas the C126W variant displays diffuse nuclear staining, indicating impaired DNA binding and altered pericentromeric localization with no evidence of a dominant-negative effect on the WT/Mutant co-transfection experiment. (Right) Electrophoretic mobility-shift assay (EMSA) using IKBS1, IKBS4, and gSat8 probes demonstrates that wild-type IKAROS binds consensus DNA sequences, whereas the C126W variant fails to bind or is markedly reduced. Together, these findings confirm that the C126W mutation disrupts IKAROS pericentromeric heterochromatin localization and DNA-binding capacity, consistent with a loss-of-function, haploinsufficiency mechanism.

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