Journal: eLife

Article Title: PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

doi: 10.7554/eLife.26109

Figure Lengend Snippet: ( A ) Schema of the human PPP1R15A 533-624 construct used. The C-terminal Maltose Binding Protein (MBP) component, which stabilizes the fusion protein, is noted. ( B) Upper panel . Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2α P to eIF2α 0 in 20 min dephosphorylation reactions constituted with eIF2α P [2 µM], PP1 [0.625 nM], G-actin [1.5 µM] and an escalating concentration of PPP1R15A 533-624 . Shown is a representative of three independent experiments performed. Lower panel : Semi-log 10 plot of the initial velocity of eIF2α P dephosphorylation as a function of PPP1R15A 533-624 concentration derived from three repeats (one shown above). The EC 50 for PPP1R15A 533-624 was calculated using the agonist fitting function on GraphPad Prism V7. ( C) Upper panel . As in ‘B’ but dephosphorylation of eIF2α P to eIF2α 0 was carried out in the presence of a fixed concentration of PPP1R15A 533-624 [50 nM] and an escalating concentration of G-actin. Shown is a representative of two independent experiments performed. Lower panel : Semi-log 10 plot of initial velocity as a function of G-actin concentration derived from two repeats (one shown above). DOI: http://dx.doi.org/10.7554/eLife.26109.004

Article Snippet: The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7.

Techniques: Construct, Binding Assay, Staining, SDS Page, De-Phosphorylation Assay, Concentration Assay, Derivative Assay

Journal: eLife

Article Title: PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

doi: 10.7554/eLife.26109

Figure Lengend Snippet: ( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2α P in a 20 min reaction, as in above, but with an escalating concentration of mutant human PPP1R15A (533-624)R578A . Shown is a representative of three independent experiments performed. The position of the mutation is provided in the schema above the gel. The plot of initial velocity as a function of PPP1R15A (533-624)R578A derived from three repeats (one shown) is below the SDS-PAGE image. The EC 50 for PPP1R15A (533-624)R578A was calculated using the agonist fitting curve in GraphPad Prism V7. ( B ) Time-course of eIF2α P dephosphorylation using a low concentration of PP1 [0.625 nM], saturating concentrations of G-actin [400 nM], and wildtype [100 nM] or mutant human PPP1R15A (533-624)R578A [100 nM in one assay and 200 nM in the two other assays]. Shown is a representative of three independent experiments performed. Below the gel is a plot of the fraction of substrate dephosphorylated as a function of time derived from three repeats (one shown). The slope of the reaction was derived by fitting the data to a linear model in GraphPad Prism V7. ( C ) As in ‘A’ above but with saturating concentration of PPP1R15A (533-624)R578A [100 nM] and escalating concentration of G-actin. Shown is a representative of three independent experiments performed. The plot of initial velocity as a function of G-actin derived from three repeats (one shown) is below the SDS-PAGE image. The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7. DOI: http://dx.doi.org/10.7554/eLife.26109.006

Article Snippet: The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7.

Techniques: Staining, SDS Page, De-Phosphorylation Assay, Concentration Assay, Mutagenesis, Derivative Assay

Journal: eLife

Article Title: PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

doi: 10.7554/eLife.26109

Figure Lengend Snippet: ( A ) Schema of the biotinylated human PPP1R15A 533-624 immobilized onto the BLI biosensor tip. ( B ) Plot of Bio-Layer Interferometry (BLI) signal as a function of time in a representative experiment (repeated three times) in which immobilized PPP1R15A 533-624 was reacted with PP1 [40 nM] in solution (blue trace). The fitting curve using ‘association then dissociation’ model in GraphPad Prism V7 is shown in red. Vertical dashed line marks the beginning of the dissociation phase. Table summarizes kinetic parameters extracted from fitting curves of three repeats of the experiment shown in left panel (mean ± standard deviation). ( C ) As in ‘B’ above, but the immobilized PPP1R15A 533-624 was first exposed to PP1 [200 nM], before being exposed to a solution of both PP1 [200 nM] and G-actin [400 nM]. Shown is a representative of an experiment repeated three times. DOI: http://dx.doi.org/10.7554/eLife.26109.007

Article Snippet: The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7.

Techniques: Standard Deviation

Journal: eLife

Article Title: PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

doi: 10.7554/eLife.26109

Figure Lengend Snippet: ( A ) Plot of Bio-Layer Interferometry (BLI) signal as a function of time in a representative experiment (repeated three times) in which immobilized wildtype and indicated mutant PPP1R15A 533-624 proteins were reacted with PP1 [40 nM] in solution (thick traces). The fitting curve using ‘association then dissociation’ model in GraphPad Prism V7 is shown in thin red line. Vertical dashed line marks the beginning of the dissociation phase. Table summarizes kinetic parameters extracted from fitting curves of three repeats of the experiment shown in left panel (mean ± standard deviation). ( B ) As in ‘A’ above, but the immobilized wildtype and mutant PPP1R15A 533-624 probes were first reacted with PP1 [200 nM], before being exposed to a solution of both PP1 [200 nM] and G-actin [400 nM]. Shown is a representative of an experiment repeated three times. DOI: http://dx.doi.org/10.7554/eLife.26109.008

Article Snippet: The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7.

Techniques: Mutagenesis, Standard Deviation

Journal: eLife

Article Title: PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz

doi: 10.7554/eLife.26109

Figure Lengend Snippet: ( A ) Coomassie-stained PhosTag-SDS-PAGE tracking the dephosphorylation of eIF2α P in 20 min reactions constituted with PP1 [0.625 nM], G-actin [1.5 µM] and an escalating concentration of human PPP1R15A 325-636 . Shown is a representative of three independent experiments performed. A schema of the human PPP1R15A 325-635 construct is shown above the gel. A semi-log 10 plot of the initial velocity of eIF2α P dephosphorylation as a function of PPP1R15A 325-636 concentration derived from three repeats of the experiment is shown below. The EC 50 for PPP1R15A 325-636 was calculated using agonist fitting function on GraphPad Prism V7. ( B ) As in ‘A’ above, but in the presence of a fixed concentration of PPP1R15A 325-636 below the EC 50 [2 nM] and escalating concentrations of Sephin1. Shown is a representative of the two independent experiments performed. Plot contains data from the two repeats. ( C ) As in ‘B’ above, but in the presence of an escalating concentrations of the PP1 active site inhibitor tautomycin (Tau). Shown is a representative of the two independent experiments performed. Plot contains data from the two repeats. ( D ) As above, triplicate reactions of eIF2α-P dephosphorylation conducted in the absence or presence of Sephin1 or the related compound, Guanabenz. ( E ) As in ‘D’ using Sephin1, salubrinal or tautomycin. Shown is a representative experiment, (of two repeats). DOI: http://dx.doi.org/10.7554/eLife.26109.010

Article Snippet: The EC 50 for G-actin was calculated using the agonist fitting curve in GraphPad Prism V7.

Techniques: Staining, SDS Page, De-Phosphorylation Assay, Concentration Assay, Construct, Derivative Assay