Figure 8

In vivo phosphorylation of PBL1 or BIK1 after flg22 treatment. Protoplasts were transfected with plasmids expressing HA-tagged BIK1 (A), PBL1 (B), or the indicated variants. After overnight expression of the proteins, the protoplasts were treated with 100 nM flg22 (10 min), harvested and subjected to western blotting with anti-HA. In vivo phosphorylation is implicated by a reduced mobility of the protein (highlighted with black arrowheads). Amido black staining of the nitrocellulose membranes was used to estimate equal loading. In (C), autophosphorylation of the immunoprecipitated kinases was used to determine if the kinase activities have been compromised by the mutations. The experiment was performed three times with similar outcome. Note that the autophosphorylation of the wild type (WT) kinases in the untreated protoplasts is variable and typically low but a weak band can be seen (indicated by asterisks). Autophosphorylation of the G70D, A97V and R172Q variants were always not visible (or lower than the wild type kinases).