A. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively. 