FIGURE 4.

Immunoblotting of membrane preparations from wild-type (WT), NuoH knock-out (KO), NuoH knock-out revertant (KO-rev), and site-specific NuoH mutants. E. coli membranes (10 μg of protein per lane) were loaded on a 15% Laemmli SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred onto nitrocellulose membranes and Western blotting was carried out using the SuperSignal West Pico system. Antibodies specific to NuoB, NuoCD, NuoE, NuoF, NuoG, NuoI, and NuoM were used. Goat anti-rabbit IgG horseradish peroxidase conjugate was used as secondary antibody for the detection of the bands. Note that mutants E157K, R286A, K303A, and R291A are not shown as they were the same as the wild-type.