eIF2 α does not accumulate in PKR clusters. (A) Representative time-lapse images of cells coexpressing mRuby-PKR and mNeon-eIF2α showing that mNeon-eIF2α does not accumulate in mRuby-PKR clusters in response to poly I:C treatment. (B) Representative immunofluorescence images showing that phosphorylated eIF2α is excluded from PKR clusters. The micrograph crops on the right show that phosphorylated eIF2α decorates the periphery of mRuby-PKR clusters. Scale bar: 10 µm. (C) Quantification of the MFI of the phosphorylated eIF2α signal in immunofluorescence analyses (mean fold-change and SEM, N = 3 experiments, n > 300; ****, P < 0.0001 unpaired Student’s t test, nonparametric). SA, sodium arsenite, positive control. (D) Representative time-lapse images showing that the fluorescently labeled PKR pseudosubstrate mNeon-K3L enters mRuby-PKR clusters. Note the ∼10-min time lag between formation of mRuby-PKR clusters and recruitment of mNeon-K3L to them. Scale bar: 10 µm.