Figure 7

Mutating Lys17 into Asp in full-length Bcl-2 largely prevents the ability of Bcl-2 to bind to the IP₃R, inhibit IP₃R-mediated Ca²⁺ release and protect against STS-induced apoptosis in COS-1 cells. (a) Lysates from 3xFLAG-Bcl-2- and 3xFLAG-Bcl-2 K/D-overexpressing COS-1 cells were used in GST pull-down assays using purified GST-Dom3. A representative western blot using anti-FLAG antibodies is shown. (b) Immunoreactive bands of at least five independent experiments were quantified using ImageJ software. Values represent normalized values relative to their binding to GST. (c–e) COS-1 cells were co-transfected with mCherry plasmids and threefold excess of either empty vector (c), 3xFLAG-Bcl-2 plasmid (d) or 3xFLAG-Bcl-2 K/D plasmid (e). Only cells expressing the mCherry plasmid were monitored as F340/F380. Fura-2 signals were calibrated to obtain [Ca²⁺] (nM). Representative Ca²⁺ traces obtained from 10 Fura-2-loaded COS-1 cells in response to ATP (1 μM) are shown. (f) Quantitative analysis of the amplitude of the ATP-induced Ca²⁺ signals in mCherry-expressing COS-1 cells transfected with either empty vector, 3xFLAG-Bcl-2 plasmid or 3xFLAG-Bcl-2 K/D plasmid obtained from six independent experiments (mean±S.E.M.). ^*Both 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D significantly reduce the peak [Ca²⁺] in response to ATP. ^$3xFLAG-Bcl-2 K/D is significantly different from 3xFLAG-Bcl-2. (g) Quantitative analysis of the area under the curve of the TG-induced Ca²⁺ signal in these cells. (h) Western-blot analysis using anti-PARP antibodies for monitoring PARP cleavage upon STS treatment (1 μM for 6 h) in 3xFLAG-vector, 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D-expressing COS-1 cells. GAPDH was used as a loading control. (i) Quantification of the immunoreactive bands of the ratio of the cleaved over the full-length PARP in the different transfected COS-1 cell populations from three independent experiments. The ratio of cleaved over full-length PARP obtained for control cells were set at 100% and the other ratios were normalized to this value. Both 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D significantly (^*) prevent STS-induced apoptosis compared with the empty-vector control, but 3xFLAG-Bcl-2 K/D is significantly ($) less potent than 3xFLAG-Bcl-2. Statistically significant differences are indicated as in Figure 5. We wish to remark that the results obtained with 3xFLAG-Bcl-2 K/D were borderline significantly (^*!) different from the empty-vector control (P=0.051)