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. Author manuscript; available in PMC: 2016 Jun 8.
Published in final edited form as: Cancer Cell. 2015 May 21;27(6):852–863. doi: 10.1016/j.ccell.2015.04.010

Figure 3. BH3 domain in Bcl2 is essential for BDA-366 regulation of Bcl2 and Bax function and Cytc release.

Figure 3

(A) 10ng of purified WT and BH3 deletion mutant (ΔBH3) Bcl2 protein was analyzed by Western blot. (B) Purified WT or ΔBH3 mutant Bcl2 in the absence or presence of Bax protein was treated with BDA-366 or BAM7, followed by IP using 6A7 Bax antibody. Conformationally changed Bax was analyzed by Western blot using Bax antibody. (C) WT and BH3 deletion mutant (ΔBH3) Bcl2 were stably expressed in H1299 cells. (D) H1299 cells expressing WT, ΔBH3 or vector-only control were treated with BDA-366 for 24h, followed by co-IP using Bcl2 BH3 specific or 6A7 antibody, respectively. (E) Isolated mitochondria from H1299 cells expressing WT, ΔBH3 or vector-only control were treated with BDA-366 in mitochondrial buffer for 30 min at 30°C. After centrifugation, Cyt c in supernatant or mitochondrial pellet was analyzed by Western blot. See also Figure S3.