(A) Western blot analysis of cerebellar extracts from 3-week-old mice incubated with the antibodies shown (left panel). The relative abundance of phospho-eIF2α (Ser51) to total eIF2α was averaged for three biological replicates (right panel). Values are shown relative to B6J. (B) The effect of Gcn2 deletion on the 146 genes upregulated or the 191 genes downregulated by 1.5 fold (padj < 0.01) in the cerebellum of B6J-Gtpbp2nmf205-/- (B6J-nmf205-/-) relative to that of B6J (left and right diagram, respectively). ATF4 target status of the 83 genes that are upregulated in the B6J-Gtpbp2nmf205-/- cerebellum relative to B6J and that also have a significant decrease in expression in the B6J-Gtpbp2nmf205-/-; Gcn2-/- cerebellum relative to B6J- Gtpbp2nmf205-/- (middle diagram). (C) Fold change of upregulated ATF4 downstream genes from B6J-Gcn2-/-, B6J-Gtpbp2nmf205-/-, and B6J-Gtpbp2nmf205-/-; Gcn2-/-mice relative to B6J expression. The 10 most upregulated genes in cerebellum of B6J-Gtpbp2nmf205-/-mice relative to that B6J are shown. Note that the increased expression of these GCN2-ATF4 downstream genes in the B6J-Gtpbp2nmf205-/- cerebellum was completely suppressed by deletion of Gcn2. Error bars = SEM. *p<0.05, **p<0.01, and ***p<0.001 (one-way ANOVA, A, C).