Figure 5. Inhibition of IPA-mediated PERK activation by GSK2606414.
(A) Reversal of IPA-mediated PERK activation ([IPA] = 0.5 and 1 μM; lanes 5 and 6; note peak in Figure 3C at these concentrations) in combination with GSK2606414 (GSK, 1 μM) was observed using immunoblotting as described in (Figure 3C). As a control, GSK (1 μM) was used to also inhibit PERK branch activation by thapsigargin (Tg; 100 nM; lane 3). (B) HEK293T cells were incubated with [35S] methionine to monitor protein translation upon addition of IPA (1 μM) or a combination of IPA (1 μM) and GSK (1 μM). The UPR was induced with Tg (100 nM) or DMSO as indicated. (C) Cotreatment of HEK293T cells with IPA + GSK. HEK293T cell viability was measured as a dose-response of IPA in combination with 1 μM of GSK (pink circles). The presence of 1 μM GSK shifted the IPA LD50 from 0.82 μM to 6.21 μM. The change in cell viability ± GSK inhibitor is overlaid on both dose responses (green bars). Cells were treated with compounds for 24 hr and cell viability was normalized to DMSO controls.
Figure 5—figure supplement 1. Quantification of [35S] incorporation.
Figure 5—figure supplement 2. The effects of GSK on HEK293T cell viability.
Figure 5—figure supplement 3. The effect of staurosporine (STS) on HEK293T cell viability in the presence of GSK PERK inhibitor.
Figure 5—figure supplement 4. Inhibition of IPA-mediated PERK activation using GSK2606414.
