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Quantification of p38 MAPK Phosphorylation of GST-FOXO3a-HIS6 Peptides

This study presents HPLC chromatograms of GST-FOXO3a-HIS6 tryptic peptides post in vitro phosphorylation with p38 MAPKs. The GST-FOXO3a-HIS6 protein was phosphorylated by p38β, p38γ, or p38δ, followed by SDS-PAGE, gel excision, trypsin digestion, and peptide separation via HPLC on a Vydac C-18 column using an acetonitrile gradient. 32P radioactivity was monitored with an online detector. This figure provides insights into the phosphorylation dynamics and peak identification in the process.

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Quantification of p38 MAPK Phosphorylation of GST-FOXO3a-HIS6 Peptides

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  1. 5000 80 50 32P radioactivity (cpm) Acetonotrile (%) 2000 30 0 0 8000 80 5000 50 32P radioactivity (cpm) Acetonotrile (%) 30 2500 0 14000 80 10000 50 32P radioactivity (cpm) Acetonotrile (%) 30 5000 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time (min) Supplementary Figure S1. HPLC chromatograms of GST-FOXO3a-HIS6 tryptic peptides following in vitro phosphorylation with p38 MAPKs GST-FOXO3a-HIS6 was phosphorylatedin vitro by p38β, p38γ or p38δ. The phosphorylated protein was then subjected to SDS-PAGE, excised from the gel, digested with trypsin and the resultant peptides separated by HPLC on a Vydac C-18 column developed with an acetonitrile gradient (broken line), as described. 32P radioactivity was detected using an online radioactivity detector (solid line). Peaks 1-5 are indicated.

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