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. 2000 Nov 28;97(25):13655–13660. doi: 10.1073/pnas.250485597

Figure 5.

Figure 5

Transcriptional inhibition of mHRT2 independent of the HRT-binding site. (A) Electrophoretic mobility shift assays were performed by using oligonucleotide probes containing a CACGTG motif and in vitro-translated HRT proteins. Specific DNA-protein complexes are indicated by an arrowhead. mHRT2 C(−), mHRT2 mutant without YQPW-TEVGAF motif; mHRT2 B(−), mHRT2 mutant lacking basic domain. (B) 10T½ cells were transfected with 5′-deletion mHRT2-luciferase constructs and Notch1 IC plasmid (150 ng each) and the effects of coexpression of mHRT2 were examined. (C) The 190-bp mHRT2-luciferase construct and Notch1 IC plasmid (150 ng each) were transfected and the effects of cotransfection of various HRT constructs were examined. Luciferase activity without Notch1 IC or HRT cotransfection was given a value of 1 in B and C. (D) The 10-kb mHRT2-luciferase construct (150 ng) and Notch1 IC and/or mHRT2 expression constructs were transfected into 10T½ cells. Trichostatin A (TSA; 300 μM) or vehicle was added to the medium 24 h after transfection and cells were incubated for 20 h. TSA treatment increased luciferase and LacZ activities in control cells with vector transfection, and the luciferase activity of cells with Notch1 IC cotransfection was given a value of 100%.