Fig 2. StCDPK3 phosphorylates NtRSG and StRSG1 in vitro.

Phosphorylation assays (PA) with ATPγP³² were performed using NtRSG-GST (A, B) or StRSG1-GST (C, D, E) as substrates and 6xHisStCDPK3 (A-D) or 6xHisStCDPK1 (E) as the enzyme source. Samples were analyzed by 10% SDS-PAGE. StCDPK3 kinase activity was previously confirmed using Syntide-2 as a positive control and GST alone as a negative control [43]. A. Upper panel: different amounts of NtRSG (lanes 1 to 6) were incubated with 50 ng of 6xHisStCDPK3 (lanes 1 to 4) in the presence of 20 μM Ca²⁺ (lanes 1 to 5). Autophosphorylation of 6xHisStCDPK3 was performed in the presence or absence of Ca²⁺ (lanes 8 and 9 respectively). Lower panel: western blot revealed with an anti-GST antibody (1:4000, α-GST). B. Phosphorylation of NtSRG by StCDPK3 in the presence of 20 μM Ca²⁺ (lanes 1 and 2) or 1 mM EGTA (lanes 3 and 4). C. Upper panel: different amounts of StRSG1 (lanes 1 to 5 and 7) were incubated with 6xHisStCDPK3 (100 ng) in the presence of 20 μM Ca²⁺ (lanes 1 to 4 and 7) or 1 mM EGTA (lane 5). Additionally StCDPK3 was autophosphorylated in the presence of 20 μM Ca²⁺ (lane 6) or 1 mM EGTA (lane 8). Middle and lower panels: western blots revealed with anti-GST (1:4000) and anti-His (1:5000, α-His) antibodies, respectively. Image J Software was used to estimate band intensities (AU = arbitrary units). D. Phosphorylation of StRSG1 (0.5 μg) by StCDPK3 (50 ng) in the presence of 20 μM Ca²⁺ (lane 1) or 1 and 5 mM EGTA (lanes 2 and 3). E. StRSG1 (2 μg) was incubated with 6xHisStCDPK1 (100 ng) in the presence of 20 μM Ca²⁺ (lane 1) or 1 mM EGTA (lane 2) (left panel). Middle and right panels: western blot with anti-His and anti-GST antibodies, respectively. The same reaction mixture was used in all PAs.