Figure 3. ISRIB induces dimerization of eIF2B in cells.
(A) HEK293T cells were treated with or without 200 nM ISRIB and clarified lysates were loaded on a 5–20% sucrose gradient and subjected to centrifugation. 13 equal-size fractions were collected, protein was precipitated and run on a SDS-PAGE gel and immunoblotted with the indicated antibodies. The red asterisk indicates a background band that cross-reacts with the eIF2B4 antibody. Sedimentation was from left to right. Gradients were calibrated (in Svedberg units, ‘S’) with ovalbumin (S = 3.5; Mr = 44 kD); aldolase (S = 7.3; Mr = 158 kD) and thyroglobulin (S = 19; Mr = 669 kD). Shown is a representative blot (N = 3). (B) HEK293T cells and lysates were treated with 200 nM ISRIB or 200 nM ISRIBinact (ISRIB-A18; Figure 3—figure supplement 1) and clarified lysates were loaded on a 5–20% sucrose gradient and subjected to centrifugation. 13 equal sized fractions were collected and fractions 6–9 were precipitated, trypsinized and subjected to mass spectrometric analysis. The sum of the normalized peptide intensity of each eIF2B subunit as well as two control proteins, eIF3a and PSMD1 in each fraction was plotted. Two biological replicates were analyzed per condition (N = 2, ±SEM). The number of peptides and peptide intensity in fractions 6–9 for all proteins identified are listed in Figure 3—source data 1. (C) Correlation coefficient (R) of the sum of the normalized peptide intensity profile through fractions 6–9 for each protein identified in the analysis with respect to eIF2B4 was plotted. The Correlation coefficient (R) of the sum of the normalized peptide intensity profile with respect to eIF2B4 of each protein identified are listed in Figure 3—source data 2.
DOI: http://dx.doi.org/10.7554/eLife.07314.008
Figure 3—figure supplement 1. Structures of ISRIB (ISRIB-A1) and ISRIBinact (ISRIB-A18).
Figure 3—figure supplement 2. Analysis of the gradients subjected to mass spectrometric analysis in Figure 3B.
