Protein Quantification in Plant Tissues

To determine FLAG-RGA, Myc-SPY and Myc-SEC protein levels in Arabidopsis or tobacco tissues, total proteins were extracted and analyzed by SDS-PAGE and immunoblotting as described^{48 (link)}. An anti-cMyc-HRP antibody (Sigma-Aldrich, Cat. A5598; 8,000× dilution) was used to detect Myc-SPY. A monoclonal anti-FLAG-HRP antibody (Sigma-Aldrich, Cat. A8592; 10,000× dilution) was used to detect FLAG-RGA. An anti-SEC antisera^{20 (link)} (2,000× dilution) and anti-rabbit IgG-HRP (Thermo-Fisher, Cat. 31462; 20,000× dilution) were used to detect Myc-SEC. Affinity-purified polyclonal anti-SPY antibodies (anti-LQKE^{49 (link)}; 6,000× dilution), and anti-rabbit IgG-HRP (same as above) were used to detect recombinant WT and mutant SPY proteins for the in vitro enzyme assays, and for Myc-SPY in the tobacco samples.