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. 2015 Oct 30;10(10):e0141968. doi: 10.1371/journal.pone.0141968

Fig 8. iCE analyses of ASK1 signaling complex interactions.

Fig 8

Cells were either pretreated with buffer only or secreted Klotho for 40 min before adding 5 μM ROT to induce oxidative stress. Clear lysates were co-IP with antibody against ASK1 and the eluted protein complex was subjected to immuno-CE fractionation as described in the text. (A) Immuno-CE profile of eluted complex of buffer-only-treated control cells originally separated within pH 4–8 (pI range 5.5–6.6 is shown here for clarity) showing overlapping protein signals (arrow) within the pI range 6.25–6.30 comprising the ASK1/14-3-3ζ/Trx complex assembly. Signals belonging to individual proteins dissociated from the triad were also found within the pH 4–8. Embedded is a software generated gel view of the complex within the specified pI range. Individual pIs of the proteins are indicated in parenthesis. (B) Profile of eluted complex from cells treated with ROT. Overlapping signals within pIs 6.29–6.30 (arrowed) are those of the 14-3-3ζ/Trx complex. ASK1 signal was undetectable in the presence of ROT. Also shown is the gel view within the pI 5.5–6.6 range. (C) Profile of eluted complex from cells pretreated with secreted Klotho prior to ROT treatment. Overlapping signals within the pI 6.12–6.15 range (arrow) are the protected triad ASK1/14-3-3ζ/Trx complex. Shifts in pI of the complex to acidic end are noticeable.