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. 2009 Aug 10;186(3):355–362. doi: 10.1083/jcb.200905153

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© 2009 Mérino et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — BH3 replacements in Bim change its binding profile. (A) Knockin strategy for the BH3 replacements (not to scale). Note that the introduced sequences do not overlap the splice sites. (B) Normal expression of the Bim mutant proteins and Bcl-2 family members in the thymi of 6–8-wk-old knockin animals. Triton X-100 lysates of 4 × 10⁶ cells were loaded per lane. The Western blots show samples from two animals per genotype. Hsp70 provided a loading control. (C) Immunoprecipitations (IPs) from thymocyte extracts of the indicated genotypes reveal the binding of WT Bim and the mutants to prosurvival relatives. (D) Test for binding of WT Bim and the mutants to Bax. HA-tagged Bim_L (L) and Bim_S (S) isoforms of each protein were overexpressed in COS-7 cells, lysates were prepared in buffer containing Triton X-100, which promotes heterodimerization, and association with endogenous Bax was tested using an anti-HA antibody–coupled affinity resin. Only WT Bim_S showed significant binding to Bax. (C and D) The asterisks indicate nonspecific bands caused by the secondary antibody. (B–D) Molecular mass is shown in kilodaltons.