Full-length AaxB proteins were detected in immunoblots of E. coli extracts using T7-Tag monoclonal antibody. A 31-kDa T7-Tag protein standard was loaded in lane S (Novagen). Lane 1 contained 112 μg extract from E. coli DEG0147 (pDG479), containing the wild-type L2/434 aaxB pseudogene fused to an amino-terminal T7-Tag. Lane 2 contained 30 μg extract from E. coli DEG0147 (pDG543) expressing the X128W variant of aaxB from strain L2/434. Lane 3 contained protein size standards (without an epitope tag). Lane 4 contained 0.7 μg affinity-purified X128W variant of AaxB from strain L2, including a small amount of cleaved β-subunit. Lane 5 contained 117 μg extract from E. coli DEG0147 (pBAD/HisA) as a negative control. Lane 6 contained 72 μg extract from E. coli DEG0147 (pDG558) expressing the epitope-tagged aaxB from strain D/UW-3. The image was saturated to detect low levels of protein expression or cleavage, and it was processed as described for Figure 2.