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. 2014 Feb;20(2):214–227. doi: 10.1261/rna.042341.113

FIGURE 3.

FIGURE 3.

E3L ZBD is required for inhibition of PKR in vitro. (A) E3L inhibition of eIF2α phosphorylation; 2.5 nM purified Flag-His6-PKR was pre-incubated with 2.5 or 25 nM purified GST (lanes 2,3), GST-E3L (lanes 4,5), or GST-E3L-Δ80 (lanes 6,7) for 10 min at room temperature, then kinase reactions were initiated by addition of 0.2 µM His6-tagged eIF2α1–200 and 0.2 mM ATP. Reactions were resolved by SDS-PAGE and subjected to immunoblot analysis using phosphospecific antibodies against Ser51 in eIF2α (top panel, eIF2α-P). The membranes were then sequentially stripped and probed using polyclonal antiserum against yeast eIF2α (second panel, eIF2α), polyclonal anti-GST antiserum (third panel), and monoclonal anti-PKR antibody (bottom panel). (B) GST-E3L-Δ80 mutation does not impair dsRNA binding in vitro. Purified GST, GST-E3L, and GST-E3L-Δ80 were incubated with poly(I:C)-agarose for 1 h at 4°C. Bound proteins were collected by centrifugation and eluted by boiling in SDS sample buffer. Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis using polyclonal anti-GST antiserum. (T) 5% of total protein used in the assay; (U) unbound (20%); (B) protein bound (20%) to the poly(I:C) agarose.