Biochemical and functional characterization of the DD/AA substitution. (A) Typical GST pull-down experiment documenting the binding of 3xFLAG-Bcl-2 in COS-7 cell lysate to GST-Domain3 and GST-Domain3DD/AA using GST as a negative control. GelCode blue–stained membrane showing the presence of GST and the GST-fusion proteins, 7.5 μg each, in the pull-down assay are shown in the top left panel. Immunoblot using an anti-FLAG antibody coupled to horseradish peroxidase to detect 3xFLAG-Bcl-2 both in the input lane and in the pull-down lanes is shown in the bottom left panel. Quantitative analysis of the binding of 3xFLAG-Bcl-2 to GST, GST-Domain3, and GST-Domain3DD/AA by densitometry (ImageJ software) is shown in the right panel. Results (mean ± SE, 4 experiments) were normalized for the binding of 3xFLAG-Bcl-2 to GST. 3xFLAG-Bcl-2 displayed significant binding to both GST-Domain3 and GST-Domain3DD/AA (P < .01). (B) SPR demonstrating the binding of both GST-domain3 and GST-domain3DD/AA with a biotin-tagged peptide corresponding to the BH4 domain of Bcl-2 (biotin-BH4-Bcl-2). Typical sensor grams illustrating the association phase of the binding of different concentrations of GST-Domain3 (top left), GST-Domain3DD/AA (top right), and GST (bottom left) to Biotin-BH4-Bcl-2 immobilized to a streptavidin-coated sensor chip. All GST-fusion proteins were dialyzed against the interaction buffer (PBS) to minimize the buffer effect. Values were corrected for background binding of the GST-fusion proteins to Biotin-BH4-Bcl-2reverse. Resonance units (R.U.) increased with increasing concentrations of GST-Domain3 (0.037-3.3μM) or GST-Domain3DD/AA (0.037-3.3μM), but not with increasing concentrations of GST (1-20μM). Quantitative analysis of the background-corrected R.U. signals plotted as mean ± SE obtained from 3 independent experiments (bottom right), in which different concentrations of GST-Domain3, GST-Domain3DD/AA, and GST were applied to the BH4-Bcl-2-coated sensor chip. The data points obtained from GST-Domain3 and GST-Domain3_DD/AA were fitted using Origin 7.0 software by a Hill equation, yielding apparent KDs of 0.37 ± 0.13μM and 0.57 ± 0.4μM, respectively. (C) Binding of IDP_DD/AA to Bcl-2 demonstrated by biotin-streptavidin pull-down of Bcl-2 from lysate of WEHI7.2 cells overexpressing wild-type Bcl-2. Cell lysate was incubated with or without the indicated peptides for 16 hours, followed by incubation with streptavidin-coated beads. Bcl-2 bound to the beads via biotin-tagged IDP_DD/AA was detected by immunoblotting. Results are representative of 3 separate experiments with the same result. (D) Disruption of Bcl-2-IP₃R interaction by IDP_DD/AA. Bcl-2 was immunoprecipitated from lysates of WEHI7.2 cells overexpressing wild-type Bcl-2. Immunoprecipitated proteins were analyzed by immunoblotting for IP₃R3 and Bcl-2. (E) IDP_DD/AA prevents the inhibition of IP₃R-mediated Ca²⁺ release by BH4-Bcl-2, a peptide corresponding to the BH4 domain Bcl-2. A typical unidirectional ⁴⁵Ca²⁺-efflux experiment showing the Ca²⁺ release induced by 3μM IP₃ from permeabilized ⁴⁵Ca²⁺-loaded wild-type MEF cells in the presence of vehicle (■), 40μM BH4-Bcl-2 peptide (●), 40μM BH4-Bcl-2 peptide and 40μM IDP_DD/AA (▴). All peptides were incubated from 4 minutes before the addition of IP₃ to 2 minutes after its addition (bars). Data points of a representative experiment, plotted as fractional loss (percentage/2 minutes) as a function of time, were obtained in duplicate and represent mean ± SD.