FIGURE 1.

Metabolic labeling combined with tandemMOAC to identify phosphorylation substrates of MKK7-MPK3/6. Twelve-day old pER8::cMYC-MKK7 seedlings were treated with ethanol as a control (-) or with 1 μM β-estradiol (+) to activate cMYC-MKK7 transgene expression. Six hours later, seedlings were harvested and ground in liquid nitrogen to a fine powder. (A) Total protein was extracted and analyzed by SDS-PAGE, western blotting analysis, and immunodetection with antibodies to examine expression of the transgene (cMYC) and phosphorylation of MPK3/6 (pTXpY). The membrane was subjected to Ponceau S staining to check equal gel loading. (B) Reciprocal ¹⁴N- and ¹⁵N-labeled Arabidopsis seedlings were ground separately and subsequently mixed in a 1:1 (w/w) ratio. Total protein was extracted, phosphoproteins enriched via Al(OH)₃-based MOAC and digested with trypsin before peptides were desalted using graphite columns. After TiO₂-based MOAC enrichment of phosphopeptides, samples were analyzed by LC-MS/MS.