Figure 5: SMBA(s) induces Bax conformational change and promotes its oligomerization.

(a) A549 cells were treated with 5 μM of SMBA1, SMBA2 or SMBA3 for 24 h. Cells were incubated with prewarmed (37 °C) growth medium containing MitoTracker (red) for 30 min. Cells were then washed with 1 × PBS, fixed, permeabilized with ice-cold methanol and acetone, blocked with 10% mouse serum and stained with mouse monoclonal 6A7 primary and FITC-conjugated anti-mouse secondary (green) antibodies. Images were merged using Openlab 3.1.5 software. Areas of co-localization are yellow. Scale bar, 10 μm. (b) A549 cells were treated with 5 μM of SMBA1, SMBA2 or SMBA3 for 24 h. A co-immunoprecipitation experiment was carried out using the 6A7 or full-length Bax antibody. Bax was analysed by western blot. (c) A549 cells were treated with SMBA1, SMBA2 or SMBA3 (5 μM) for 24 h. After treatment, mitochondria were isolated, followed by crosslinking using BMH. Bax was analysed by western blot. (d) First, mitochondria were isolated from A549 cells and then treated with SMBA1, SMBA2 or SMBA3 (5 μM) in crosslinking buffer for 30 min at 30 °C, followed by crosslinking using BMH. Bax was analysed by western blot (full blots can be found in Supplementary Fig. 9).