B. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the "0 min" controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex. 