Figure 3
The central, modulatory domain of IP3R1 binds to immobilized biotin-BH4-Bcl-2, but not to biotin-BH4-Bcl-Xl, in SPR experiments. (a–c) Sensorgrams are obtained after background correction for binding to the respective scrambled versions of the biotinylated BH4-domain peptides. The association phase is shown upon addition of different concentrations of purified GST-Dom3 to biotin-BH4-Bcl-2 (a) or biotin-BH4-Bcl-Xl (b). A typical experiment is shown. (c) The quantitative analysis of the binding properties of GST-Dom3 to either biotin-BH4-Bcl-2 or biotin-BH4-Bcl-Xl is shown. Values were obtained from three independent experiments and are plotted as mean±S.E.M. (d) Sensorgrams representing the association phase of different concentrations of GST-Dom3 for the binding to either biotin-BH4-Bcl-2 or biotin-BH4-Bcl-2BIND. All curves are background corrected for binding to the respective scrambled peptides. (e) Sensorgrams representing the binding of purified GST-Dom3 (3.3 μM) obtained from IP3R1, IP3R2 and IP3R3 to biotin-BH4-Bcl-2. GST (3.3 μM) was included as a control. (f) Quantitative analysis of the binding of purified GST and GST-Dom3 obtained from IP3R1, IP3R2 and IP3R3 (all at 3.3 μM) to biotin-BH4-Bcl-2. Values were obtained from three independent experiments and are plotted as mean±S.E.M. (g) A representative GelCode Blue-stained 4–12% Bis-Tris NUPAGE gel run in MOPS-SDS buffer showing the quality of the purified GST and GST-Dom3 derived from IP3R1, IP3R2 and IP3R3 used in these SPR experiments. The upper arrow indicates the full-length GST-IP3R-fusion proteins, whereas the lower arrow indicates parental GST
