Table 2. Specific binding of 1–225 type 1 or 3 to immobilized GST–226–604 type 1 or 3 in the presence of 2-mercaptoethanol, NEM or thimerosal.
Purified 1–225 proteins were incubated with immobilized GST–226–604 fusion proteins in their respective interaction buffer, containing 2-mercaptoethanol (1 mM), NEM (300 μM) or thimerosal (100 μM). After washing and elution, the proteins (the equivalent of 125 or 200 ng) were separated on NuPAGE® 4–12% Bis-Tris gels and analysed by Western blotting using Rbt475 (1:3000). Immunoreactive bands were quantified by using ImageQuant 5.2 software and the binding efficiency was calculated against 50 ng of input protein (%). Specific binding was determined by subtracting the binding to immobilized GST in the presence of 2-mercaptoethanol, NEM or thimerosal. Results are the means±S.D. for at least four independent experiments. Abbreviation: NS, not significant.
| Specific binding (%) (means±S.D.) | ||
|---|---|---|
| GST–226–604 IP3R1 | GST–226–604 IP3R3 | |
| 1–225 protein IP3R1 | ||
| 2-Mercaptoethanol | 5±2 | NS |
| NEM | 7±2 | NS |
| Thimerosal | 33±6 | 6±2 |
| 1–225 protein IP3R3 | ||
| 2-Mercaptoethanol | NS | NS |
| NEM | NS | NS |
| Thimerosal | 4±2% | NS |