In vitro kinase assays for STPKs.

The purified STPKs (Pkn A, B, D, J, L, G and K) were incubated in PIPES buffer (100 mM PIPES, pH 7.0, 80 mM NaCl and 20 mM MgCl₂) containing 1-2μCi of [γ-³²P] ATP for 10-45 minutes at 25°C. Autophosphorylation assays of PknE, PknF, and PknH were performed in buffer containing 25 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 2mM MnCl2, and 1 mM DTT for 30 min at 37°C. The reaction was stopped by adding of 5X SDS sample loading buffer. To check the effect of NU-6027 (dissolved in DMSO), variable concentration (0.01-100µM) of NU-6027 was added in the reaction mixture and incubated for the same duration as control for a particular STPK. In control set DMSO was used instead of NU-6027. The effect of NU-6027 (50 & 100 µM) was checked against PknA, B, D, G and K. However, the autophosphorylation activity inhibition of PknE, F, H, J and L was performed against 50mM concentration of NU-6027. Proteins were resolved on 12%SDS-PAGE. After electrophoresis, the gel was wrapped in cellophane sheet and exposed to a phosphor imaging plate for 12 hours followed by scanning with Typhoon 9210 imager. The gel was then stained with Commassie R₂₅₀ to visualize the protein bands.  Image J was used to calculate the intensity of autophosphorylation of Pkns (with and without inhibitor) and Ic50 was calculated using GraphPad Prism7.