-Secretase Cleavage and Nuclear Localization of ErbB-4 Receptor Tyrosine Kinase Chang-Yuan Ni, M. Paul Murphy, Todd E. Golde, Graham Carpenter

Supplementary Material

Supplemental Figure 1. Analysis of the transactivation potential of the ErbB-4 cytoplasmic domain fragments in the GAL4 system (1). The GAL4 DNA binding domain (GAL4BD, residues 1-141) was fused to the ErbB-4 cytoplasmic domain (ErbB-4CD, residues 676-1308), the ErbB-4 kinase domain (ErbB-4KD, residues 676-988), or the ErbB-4 COOH-terminal domain (ErbB-4CT, residues 988-1308). As a positive control, a fusion of GAL4BD with the transcription factor E2F1 was used. Each of these fusion constructs plus GAL4BD was co-transfected into COS7 cells with a reporter plasmid encoding firefly luciferase controlled by a GAL4-responsive promoter. Also, a reporter plasmid encoding the Rellina luciferase controlled by a constitutive promoter was co-transfected to normalize transfection efficiencies. The Qiagen PolyFect kit was used for transfection and ratio of transfected constructs were 12:2:1 for GAL4 fusion protein (or GAL4BD) : Firefly luciferase : Renilla luciferase plasmids. Approximately 48 hrs after transfection, the cells were lysed and assayed using the Promega Dual- Luciferase Reporter Assay System.

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Supplemental Figure 2. Intracellular localization of ErbB-4 cytoplasmic domain fragments. Constructs of GFP with the ErbB-4 kinase domain (GFP~ErbB-4KD, residues 676-988, approximately 64kDa) or the ErbB-4 COOH-terminal domain (GFP~ErbB-4CT, residues 988-1308, approximately 63kDa) were prepared using the vectors PEGFP-C1 and PEGFP-N2 from Clontech. The transfection was done with Effectene from Qiagen. Each construct was then expressed in COS7 cells and localized by confocal microscopy, as described in Figure 4. Bar, 25m, applies to each image.

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Reference

1. M. Vecchi et al., J. Cell Biol. 153, 1511 (2001).