Ac

eto

no

trile (%

)

0

30

50

80

Ac

eto

no

trile (%

)

0

30

50

80

32P

ra

dio

ac

tiv

ity

(c

pm

)

50000

16000

32P

ra

dio

ac

tiv

ity

(c

pm

) 10000

14000

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 phosphorylated in 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).

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Time (min)

12000

8000600040002000

0

40000

30000

20000

0

10000

Ho et al Fig. S4