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Understanding the Role of PknJ in Mycobacterium tuberculosis: Biochemical Characterization and Identification of Novel Substrate Pyruvate Kinase A

Figure 2

In vitro kinase assay of PknJ, Phosphoamino acid analysis and Immunoblotting of PknJ-KD and activation loop mutants.

(A) Graph showing time-dependent autophosphorylation of PknJ-KD. After in vitro kinase assay with [γ-32P]ATP, gel was analyzed by PhosphorImaging and counts were calculated with MultiGauge (FujiFilm). Counts obtained after 30 min of reaction were taken as 100% signal and relative phosphorylation was estimated. Experiment was performed twice and the results indicate average of the two. (B) Analysis of phosphoamino acid content of autophosphorylated PknJ-KD. Amino acid standards, phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) were added in the radiolabeled sample and visualized by ninhydrin staining (left panel) prior to autoradiography (right panel). The labeled pThr, pSer and their corresponding standards are encircled. (C) In vitro autophosphorylation of PknJ-KD (1 µg) and phosphotransfer on 5 µg Myelin basic protein (MyBP). Autophosphorylation deficient mutant PknJ-KD-K43A was used as negative control for auto- and transphosphorylation. The reactions were run on 12% SDS-PAGE and gel was autoradiographed after drying. As shown, transphosphorylation on MyBP was visible only in the presence of PknJ-KD, lane2. (D, E, F) 2 µg of in vitro phosphorylated native kinase along with its activation loop mutants (as indicated) were resolved on 10% SDS-PAGE, transferred onto nitrocellulose membrane and probed with anti-phosphothreonine antibody as described in experimental procedures. Autoradiograms are shown.

Figure 2