FIG. 7.

Pull-down assay of XpsN proteins with alanine substitutions, with XpsL-XpsM(His₆) immobilized on Ni-NTA resin. The XpsL-XpsM(His₆) complex prepared as Triton X-100 extract from XC17433(pRT40) was immobilized on Ni-NTA resin. After a thorough wash, the resin was mixed with the Triton X-100 extract of the membrane proteins prepared from XC17433 supplemented with plasmid that expresses the wild-type XpsN(pNC2), mutated XpsNE67A(pCPPE67A), or XpsNR78A(pCPPR78A). After being washed, the bound proteins were eluted with buffer containing 250 mM imidazole. T, total Triton X-100 extract of membrane proteins; E, bound proteins eluted from the resin at five times the concentration of T. For immunoblotting, anti-XpsN antibody (top), anti-XpsL antibody (middle), and anti-XpsM antibody (bottom) were used.