Figure 3.

Western blot demonstrating cross-linking of S143C/S336C in native vesicles. Native membrane vesicles (see Experimental Procedures) containing OxlT with the S143C/S336C mutations and a tandem Factor Xa cleavage site were treated with 200 μM copper(II)(1,10-phenanthroline)₃(CuPhe), 200 μM MTS-1-MTS (M1M), or 200 μM MTS-2-MTS (M2M) for 10 min and immediately quenched with 5 mM NEM and 10 mM EDTA. Washed vesicles were processed for western blotting and probed with a C-terminal polyhistidine antibody. Xlink indicates lanes with the addition of the indicated cross-linking agents; Factor Xa indicates lanes where the central loop of OxlT has been cleaved; DTT indicates lanes where DTT is used to reduce disulfides after Factor Xa cleavage. The positions of the covalently intact protein (OxlT), the C-terminal OxlT fragment (C-ter), and OxlT oligomer (OxlTn) are indicated.