Figure 5. Identification of a mutant of R15B defective in substrate binding.

(A) Sequence conservation of R15B^414–639. The residues are colored according to ConSurf⁴¹ conservation scores from cyan (variable) to burgundy (conserved). The consensus secondary structure, predicted using Jpred⁴² and PsiPred,³⁶ is shown below the corresponding sequence. The secondary structural elements are denoted as follows: rectangle, helix; line, random coil. The substitutions of the residues targeted for mutagenesis are shown on the top with different mutants shown in different colors.

(B and C) R15B^411–613 (B) or full-length (C) wild-type (WT) or mutants were transfected into HEK 293T cells (input) and immunoprecipitated using anti-FLAG M2 magnetic beads (FLAG-IP). Immunoprecipitated complexes were eluted and analyzed on a 4%–12% bis Tris Plus gel. Proteins were detected by immunoblotting with FLAG, eIF2α, PP1, and vinculin antibodies. Representative results of at least 3 experiments are shown.

(D) Activity of transfected R15B full-length WT or H1A assessed by decreased levels of P-eIF2α. Proteins were detected by immunoblotting with R15B (R15B-4D11), P-eIF2α, eIF2α, PP1, and vinculin antibodies. Representative results of at least 3 experiments are shown.

(E) Quantifications of P-eIF2α from 3 experiments such as the one shown in (D). Data are mean ± SD. (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA.