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. 2017 Apr 11;8:522. doi: 10.3389/fpls.2017.00522

FIGURE 4.

FIGURE 4

Phosphorylation of TaeNAD-GAPDH versions. (A,B) In vitro phosphorylation of NAD-GAPDH wild type and mutant S66A (A), and NAD-GAPDH mutant S124A and S205A (B), with plant extracts under kinases phosphorylation conditions for (1) WPK4, (2) SOS2, (5) CKII, (6) Tsl, and (7) CDPK. Incorporation of [32P]ATP was detected by autoradiography (upper image) of SDS-PAGE gel stained with Coomassie Blue (bottom image). (#) Recombinant proteins in the absence of the plant extract. (C,D) In vitro phosphorylation of all the NAD-GAPDH versions by the purified SnRK1 resolved by SDS-PAGE and revealed by storing phospho-screen exposure and scanning with the TyphoonTM system (C) or stained with Coomassie Blue (D). Numbers show the NAD-GAPDH versions: WT in the absence of SnRK1 (1); WT oxidized (2) or reduced (3) in the presence of SnRK1; S66A oxidized (4) or reduced (5) in the presence of SnRK1; S124A oxidized (6) or reduced (7) in the presence of SnRK1; S205A oxidized (8) or reduced (9) in the presence of SnRK1. Arrows indicate the migration of NAD-GAPDH. All the assays were also perform in the absence of any recombinant protein to identify the background phosphorylation.