Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

Figure 2

Phosphorylation activity and immunoprecipitation of MAP PknG. (a) In vitro phosphorylation of MBP by MAP PknG. Recombinant his-tagged PknG was purified and incubated with [γ- ³²P] ATP and MBP. Samples were separated by SDS-12% PAGE and stained with Coomassie blue followed by visualization by autoradiography. Lanes: 1, recombinant PknG; 2, MBP; 3, recombinant PknG + MBP. Molecular mass markers are indicated on the left in kDa. (b) The immunoprecipitants of infected and noninfected THP-1 cells were resolved by SDS-10% PAGE, electroblotted onto a nitrocellulose membrane, and exposed to rabbit anti-PknG antibodies. Lanes: 1, THP-1 cell-free lysate; 2, BSA (negative control); 3, recombinant PknG; 4, THP-1 cell lysate; 5, MAP-infected THP-1 lysate. Molecular mass markers in kDa; arrow points to the whole PknG.

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