Figure S1. Schematic representation of the plant proteome separation using the PF2D system. The collected fractions are transferred to polyvinylidene difluoride (PVDF) membranes that were hybridized with antibodies against different post-translational modifications (PTMs) and detected by chemiluminescence. The positive immunoreactive proteins were identified by mass spectrometry (MS) or by liquid chromatography MS/MS.

Figure S2. Identification of threonine phosphorylated proteins. (A) First-dimension fractions number 1, 2, and 3 were resolved in the second dimension and then transferred to polyvinylidene difluoride (PVDF) membrane by dot blot. These membranes were analyzed using anti-phosphothreonine (clone H2) (Santa Cruz Biotechnology, CA, USA). A subset of anti-phosphothreonine immunoreactive spots from the second dimension were analyzed by mass spectrometry (MS). The signal labeled as ``a'' and ``b'' corresponded to the fractions that identified the protein At1g54010 in both cases. (B) Identification of phosphorylated proteins by MS. A subset of the anti-phosphothreonine immunoreactivated spots were analyzed by MS. (*) Ions score is -10*Log(P), where P is the probability that the observed match is a random event (MASCOT program). Phosphorylation consensus sequences were obtained with the Netphos2.0 program ( tool. This analysis revealed, with a high score, several threonine residues that may be the phosphorylation sites in these proteins. Interestingly, one of the identified proteins, CRUCIFERIN3 (CER3), has already been described as a phospho-protein (Sugiyama et al., 2008).

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