Fig. 4.

In vitro kinase activity of native recombinant GST1-tagged proteins. (A) Phosphorylation of SLAC1 termini (NT and CT) by OST1 was tested by using radio-labeled [γ³²] ATP (Left: Coomassie stained SDS PAGE; Right: radioautogram of the gel; the presence of proteins in the reaction assay was indicated by +). Only SLAC1 NT was phosphorylated by OST1. In contrast to OST1 WT, the OST1 mutant D140A did not phosphorylate SLAC1 NT. Arrowheads indicate the position of recombinant proteins. (B) In vitro kinase assays with native recombinant proteins revealed that OST1 activity was prevented by ABI1 and the ATP analogue ATPγS. When SLAC1 NT was phosphorylated before adding ABI1 together with ATPγS (indicated by the black box, lane 4) we could not detect any dephosphorylation activity of ABI1 within 45 min of incubation at RT. The molecular weight of the protein ladder is indicated in kDa.