Figure 3.

MBD domain of 4.1R interacts with the β-catenin armadillo domain repeats 1–2. A, ³⁵S-labeled in vitro–translated full-length β-catenin (β-cat) was incubated with GST–4.1R¹³⁵ and GST–4.1R⁸⁰ (left) or its individual domains (right) bound to GSH-Sepharose beads and analyzed for the presence of β-catenin with the GST fusions in GST-pulldown analyses. Upper panel, Coomassie Blue–stained GST fusions. Lower panel, GST-pulldown analyzed for the presence of β-catenin with GST–4.1R fusions. B, ³⁵S-labeled in vitro–translated full-length β-catenin or its individual NTD, ARM, or CTD domains were incubated with GST–MBD bound to GSH-Sepharose beads and examined for the presence of β-catenin or its domain(s) in each input and with GST–MBD fusions (bead). C, analyses of the repeats within the armadillo domain responsible for 4.1R–MBD interactions in yeast two-hybrid assays. 4.1R–MBD fused with pGBKT7 was co-transformed with the full-length armadillo domain fused with Gal4 DNA-AD in pGADT7 (1–12/pGADT7) or its repeat-deletion constructs (1–10/pGADT7, 1–8/pGADT7, 1–4/pGADT7, 1–2/pGADT7, 3–12/pGADT7, and 5–12/pGADT7) and analyzed for β-gal activity. Transformation of pGBKT7 or pGADT7 vector without fusion protein served as a negative control. Co-transformation of p53/pGBKT7 and large T-antigen/pGADT7 served as a positive control. D, interaction of 4.1R–MBD and the armadillo domain repeats was confirmed in GST-pulldown analyses. ³⁵S-Labeled in vitro–translated full-length or its repeat-deletion β-catenin armadillo domains were incubated with GST–MBD bound to GSH-Sepharose beads and analyzed for the presence of β-catenin repeats with the GST–MBD fusions (bead). Molecular mass markers (kDa) are provided at the left of each blot.