Table 1. The luminescence-based mammalian interactome assay (LUMIER) detects reductions in subsets of Smad3 protein interactions due to mutations in Smad3 or the absence of Smad3 phosphorylation.
| Smad3 (WT or mutant) | Percent binding relative to the binding by phosphorylated wild-type Smad3 | |||
| Smad4 | SARA | Smurf2 | Ski | |
| FL | aa665–721 | aa252–330 | aa2–56 | |
| Smad3 WT+Alk5 | 100 | 100 | 100 | 100 |
| Smad3 WT, No Alk5 | 6 (1) | 78 (26) | Nd | nd |
| D408H | 3 (1) | 125 (45) | 23 (12) | 3 (1) |
| Del-PPGY (182–185) | 120 (15) | 129 (16) | 3 (1) | 160 (50) |
| Y297A | 181 (42) | 8 (3) | 119 (20) | 1 (1) |
| QPSMT/SE * | 132 (18) | 61 (26) | 86 (29) | 4 (1) |
Binding between wild-type or mutant Renilla-Smad3 fusion proteins and four different Flag epitope-tagged Smad-binding proteins was quantified by pull-down of protein complexes from cell lysates in anti-Flag antibody-coated 96-well plates and measuring the amount of luciferase activity per well in a luminometer. The luciferase counts were normalized to the number of counts recovered with phosphorylated wild-type Smad3 (100%). All of the Smad3 mutant proteins were co-expressed with the constitutively active Alk5. The average of the data from at least four independent transfections is shown. Total Renilla luciferase activity was measured in each transfected cell lysate as a surrogate measure for the level of Renilla-Smad fusion protein; data is only reported for experiments where the level of each Renilla-Smad3 mutant was within two-fold of the level of the Renilla-Smad3 wild type protein in the same experiment. FL indicates that the full-length Smad4 protein was used. The amino acids comprising the SARA [32], Smurf2 [52] and Ski [22] Smad3-binding motifs that were expressed on the Flag-epitope tagged thioredoxin scaffold are indicated below each gene. The reduced binding interactions of the Smad3 mutants D408H and Y297A to the Ski Smad-binding domain are consistent with previous studies using different assay methods [22]. Standard deviations are indicated in parenthesis. Not determined (nd).
*The Smad3 QPSMT/SE mutant has residues 252–256 and 266–267 changed to the Smad1 equivalent amino acids (STSVL/NKN) [47].