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. 2017 Feb 15;7:42724. doi: 10.1038/srep42724

Figure 4. HpaM interacts with HrcC and HrcJ.

Figure 4

(A) Bacterial two-hybrid assays. The BacterioMatch II two-hybrid system was used to test the interaction between HpaM and HrcC or HrcJ. The reporter strain XL1-Blue MRF′ harboring different plasmid pairs was grown on no selective plates and double-selective indicator plates containing 5 mM 3-amino-1,2,4-triazole (3-AT) and 12.5 μg ml−1 streptomycin, respectively. Protein-protein interaction activated the expression of the genes HIS3 and addA in the reporter strain, resulting in resistance to 3-AT and streptomycin. (B) Pull-down assays. His6-tagged fusion proteins were overexpressed and purified. Streptavidin sepharose beads were used to immobilize biotinylated His6-HpaMLN22, His6-HrcC34–370 or His6-HrcJ22–206, the potential prey protein was mixed with the bait protein and incubated. After elution, samples were separated on 12% SDS-PAGE and visualized by coomassie blue staining. Lanes: 1, biotinylated bait protein His6-HpaMLN22; 2, pull-down of His6-HrcC34–370 by His6-HpaMLN22; 3, bait protein His6-HpaMLN22 mixed with protein His6-HpaR1(negative control); 4, pull-down of protein His6-HrcJ22–206 by His6-HpaMLN22; 5, biotinylated bait protein His6-HrcC34–370; 6, pull-down of protein His6-HrcJ22–206 by His6-HrcC34–370; 7, pull-down of protein His6-HpaMLN22 by His6-HrcC34–370; 8, bait protein His6-HrcC34–370 mixed with protein His6-HpaR1(negative control); 9, biotinylated bait protein His6-HrcJ22–206; 10, pull-down of protein His6-HrcC34–370 by His6-HrcJ22–206; 11, pull-down of protein His6-HpaMLN22 by His6-HrcJ22–206; 12, pull-down of truncated protein His6-HpaMLN180–225 by His6-HrcC34–370; 13, biotinylated His6-HrcC34–370 was mixed with protein His6-HpaR1 (negative control); 14, pull-down of truncated protein His6-HpaMLN180–320 by His6-HrcJ22–206; M, molecular mass marker.