Figure 5.

Dominant‐negative function of adult T‐cell leukemia (ATL)‐type Helios isoforms. (a) DNA‐binding activities of wild‐type Helios or Ikaros and ATL‐type Helios proteins. Each FLAG‐tagged Helios or Ikaros isoforms were ectopically expressed in 293T cells and their nuclear extracts were subjected to EMSA with a [γ‐³²P]‐labeled Hes1 promoter probe. Oct‐1 probe was used as an internal control. Arrowheads indicate Helios or Ikaros complexes. *Non‐specific bands. Hel‐v1, Hel‐variant 1; Hel‐v2, Hel‐variant 2. (b) Results of supershift assays. Anti‐FLAG (0, 0.5, 1 μg) or control IgG (1 μg) antibodies were added to each nuclear extract prior to electrophoresis. The black and white arrowheads indicate the supershifted bands of Ik‐1 and Hel‐1, respectively. (c) Antagonistic effects of Hel‐5 on DNA‐binding of Ik‐1 tested by EMSA. The molar ratios of Ik‐1 to Hel‐5 plasmids are 1:1, 1:4, and 1:8. Expression levels of FLAG‐Ik‐1 and HA‐Hel‐5 were assessed by immunoblotting. The arrowheads indicate the Ik‐1 specific band. AP‐1 probe was used as an internal control. WB, western blot. (d) Transcriptional suppression activities of various Helios or Ikaros isoforms tested by Hes1 promoter‐luciferase reporter systems (n = 3, mean ± SD). Basal Hes1 promoter activity was defined as firefly/renilla ratio, and suppression activities of Helios or Ikaros are relatively presented. Statistical significance was evaluated by unpaired Student's t‐test (*P < 0.05; **P < 0.01). (e) Inhibitory function of Hel‐5 against Ik‐1 and Hel‐1 tested by Hes1 promoter assay (n = 3, mean ± SD). The molar ratios of Ik‐1 or Hel‐1 to Hel‐5 plasmids are 1:1, 1:2, and 1:3. Relative luciferase activities were defined as firefly/renilla ratio.