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HPLC Analysis of GST-FOXO3a-HIS6 Phosphorylation by JNKs with 32P Radioactivity Detection

This supplementary figure (Figure S4) presents HPLC chromatograms of GST-FOXO3a-HIS6 tryptic peptides after in vitro phosphorylation with JNK1α1 and JNK2α2. Phosphorylated proteins were analyzed through SDS-PAGE, excised, and digested with trypsin. Resulting peptides were separated using a Vydac C-18 column with an acetonitrile gradient. The detection of 32P radioactivity was performed with an online detector. Key peaks match those identified earlier with p38α, demonstrating consistent phosphorylation patterns.

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HPLC Analysis of GST-FOXO3a-HIS6 Phosphorylation by JNKs with 32P Radioactivity Detection

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  1. 16000 80 14000 12000 10000 50 32P radioactivity (cpm) 8000 6000 30 4000 Acetonotrile (%) 2000 0 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 50000 Time (min) 80 40000 30000 50 20000 32P radioactivity (cpm) 30 Acetonotrile (%) 10000 0 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time (min) Supplementary Figure S4. HPLC chromatograms of GST-FOXO3a-HIS6 tryptic peptides following in vitro phosphorylation with JNKs. GST-FOXO3a-HIS6 was phosphorylatedin vitro by JNK1α1 or JNK2α2. The phosphorylated protein was 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 and 2 are indicated and were found to be the same as those identified in peaks 1 & 2 following phosphorylation of FOXO3a with p38α (Figures S2 and S3). Ho et al Fig. S4

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