FIG. 7.

Limited trypsin proteolysis of membranes containing Wzz proteins and the double-replacement mutants with mutations in regions I, II, and III. Forty micrograms of total membranes was incubated at 35°C with 30 μg/ml trypsin for 2 or 16 h. Untreated samples were also incubated for 16 h. Lane M contained the broad-range prestained SDS-PAGE standard (Bio-Rad). Asterisks indicate Wzz products, and the arrow indicates undigested Wzz. Samples were separated on an 18% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane, and the blot was reacted with affinity-purified rabbit polyclonal anti-WzzPL and developed by fluorescence using IRDye800CW anti-rabbit IgG antibodies.