SAHBs were synthesized using our established method46 (link),47 (link) and recombinant BAX for NMR and biochemical analyses was generated as previously described33 (link),35 (link). Samples for HSQC and PRE NMR contained uniformly 15N-labeled BAX at 0.2 mM prepared in 10 mM sodium acetate solution at pH 6.0 with up to a 1:1 molar ratio of SAHB. NMR spectra were acquired at 32°C on Bruker 600 and 800 MHz spectrometers, and then processed and analyzed as described in the Full Methods. To evaluate BIM SAHB-induced BAX activation, four in vitro assays were performed. The oligomerization assay employed freshly purified monomeric BAX in combination with BIM SAHB at the indicated ratios and incubation durations followed by size-exclusion chromatography to quantify monomeric vs. oligomeric BAX. The BAX conformational change assay also employed the indicated BIM SAHB:BAX mixtures, which were exposed to the conformation-specific 6A7 anti-BAX antibody, followed by immunoprecipitation and BAX Western analysis to monitor the proportion of activated conformer of BAX upon BIM SAHB exposure. To determine if the BIM SAHB-induced BAX conformational change reflected functional activation of its release activity, we conducted liposomal and mitochondrial release assays as previously described33 (link),48 (link) and using the indicated doses and constructs of BIM SAHB and BAX. For cellular studies, DKO MEFs were reconstituted with BAX by retroviral transduction of BAX-IRES-GFP as previously reported7 (link),13 (link) and as described in the Full Methods. BAX or BAXK21E-reconstituted DKO MEFs were exposed to either BIM SAHBs or staurosporine, and cell death quantified over time by annexin-V-Cy3 staining followed by flow cytometric analysis.