Fig. 2.

Phosphorylation of the _XJTGB1 protein in vitro and in vivo. (A) Coomasie Brilliant Blue (CBB) staining of recombinant _XJTGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified _XJTGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ-³²P]ATP or [γ-³²P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking _XJTGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the _XJTGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of _XJTGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking _XJRNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the _XJTGB1 protein. (D) In vivo phosphorylation of _XJTGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C. (This figure is available in colour at JXB online.)