FIG. 4.

Western immunoblot assessing in vivo binding of wild-type (WT) and mutant MexA proteins to MexB-His₆. Cell envelopes of P. aeruginosa strain K2275 carrying plasmid pDN25 (pMMB206:: mexB-6his) (lanes 1 and 2) or pMMB206 (lanes 3 and 4) and pRK415 derivatives expressing the indicated MexA proteins were extracted with Triton X-100 and applied to Ni-NTA. Triton X-100-soluble cell envelope extracts (1 μl) applied to Ni-NTA (lanes 1 and 3) and DTT-treated elution fractions (10 μl) recovered from Ni-NTA (lanes 2 and 4) were immunoblotted and developed with antibodies to MexA (A) and MexB (B). See Table 1 for designations of the pRK415 derivatives expressing the indicated MexA proteins.