G 4510

For GST pull down assays, GST-Smad7 and 6XHis-β-catenin fusion proteins were produced in bacteria using standard protocol. Briefly, GST-Smad7- and 6XHis-β-catenin-expressing cells were sonicated and the proteins were purified with glutathione-agarose beads (Sigma-Aldrich G-4510). Protein concentrations were estimated by SDS-polyacrylamide gel electrophoresis and following Coomassie blue staining, using BSA for comparative estimation. GST-β-catenin fragments corresponding to aa (full length (FL), 1–100, 120–683, 422–683, 575–696, 575–683, 1–574) were utilized for mapping study. Five micrograms of GST FL Smad7, β-catenin (or the molar equivalent of the smaller GST, GST-β-catenin fragments), and 25 µl glutathione-agarose beads (50% slurry) were incubated in 600 µl NETN buffer (100 mM NaCl, 20 mM Tris-HCl (pH 8) 0.5 mM EDTA, 0.5% (vol/vol) NP-40) overnight at 4 °C. In experiments where GST-β-catenin was used to co-precipitate Smad7, GST-Smad7 was thrombin (GE Healthcare-27-0846-01) digested to yield the immune-complex without the GST tag (10 µl thrombin per mg fusion protein was incubated at room temperature for 17 h), and eluates were analyzed by western blotting.

Putative glutathione S-transferases from AX4 cells lysate were isolated with glutathione-agarose beads (G-4510, Sigma-Aldrich) using affinity chromatography protocols that were supplied by the manufacturer. Isolated protein species were resolved by SDS-PAGE analysis.