FIGURE 6.

Complex formation of the autophagy regulator Beclin-1 with Bcl-2 and Vps34 in IP₃R wild-type and TKO cells. A, levels of Bcl-2 and Beclin-1 in wild-type (WT) and TKO DT40 cell lysates (30 μg) were measured by immunoblotting. B, WT and TKO DT40 cell lysates were immunoprecipitated (IP) with a monoclonal Bcl-2 Ab, and the immunoprecipitates were probed by immunoblotting for the presence of Beclin-1. Immunoprecipitation and immunoblotting were carried out according to the instructions for the TrueBlot kit (eBiosciences, San Diego), which minimizes detection of the IgG band that runs just below the Beclin-1 band. A sample containing buffer alone and Bcl-2 mAb was used to verify the effectiveness of the method. C, Abs to IP₃R and Beclin-1 were used to immunoprecipitate 0.5 mg of WT and TKO DT40 cell lysate. The immunoprecipitates were probed by immunoblotting for the presence of Vps34. All data are representative of 2 or 3 experiments.