Fig. 3. No interaction between Bcl–2 and Flag-tagged Bcl–2 in R6 extracts. (A) α–Flag/M5 (lanes a–c) or α–mBcl–2/27–6 (lane d) immunoprecipitates from radiolabeled R6 cell extracts harboring either the transfer vector (pMV12) (lane a), Flag–mBcl–2 (R6-Flag–Bcl–2mix) (lane b), mBcl–2 (R6-Bcl–2#9) (lane d) or both (R6-Bcl–2/Flag–Bcl–2#7) (lane c). Note the absence of a band at 26 kDa in lane c, indicating that 26 kDa mBcl–2 does not co-precipitate with 27 kDa Flag–mBcl–2. (B) α–mBcl–2/10C4 Western blot of α–Flag/M5 immunoprecipitates from unlabeled extracts of R6 cells overexpressing Flag–mBcl–2 (R6-Flag–Bcl–2mix) (lanes a and e) or mBcl–2 (R6-Bcl–2#9) (lanes c and g) or both (R6-Bcl–2/Flag–Bcl–2#7) (lanes b, f and i) before (– stress) and after treatment with 1 μM MG132 (+ MG132) or depletion of serum for 48 h (– serum). Total extracts from R6-Bcl–2/Flag–Bcl–2#7 cells in the presence or absence of serum are shown in lanes d and h, respectively. Note that neither lane b nor lanes f and i contain co-precipitating 26 kDa exo mBcl–2 although it is clearly present in the total extracts (lanes d and h). (C) α–hBcl–2/100 Western blot of an extract (lane a) or an α–Flag/M5 immunoprecipitate of this extract (lane b) prepared from HEK cells co-transfected with Flag–hBcl–2 and hBcl–2. Note that the human forms of Bcl–2 also do not interact.