Figure 5. Collapsing the Golgi apparatus on to the ER restores cleavage of 3xFLAG-ATF6α in the presence of Ceapins.
(A) 293 T-REx cells stably expressing doxycycline inducible 3xFLAG-ATF6α were treated either with vehicle (ethanol) or Brefeldin A (5 μg/mL BFA) in the absence or presence of increasing concentrations of either S1P inhibitor (Pf-429242) or active (Ceapin-A1, Ceapin-A3) or inactive (Ceapin-A5) Ceapin analogs for one hour prior to harvesting lysates for Western Blot analysis of 3xFLAG-ATF6α. (B) 293 T-REx cells stably expressing doxycycline inducible 3xFLAG-ATF6α were treated either with vehicle (DMSO) or ER stressor (100 nM Tg) in the absence or presence of increasing concentrations of either S1P inhibitor (Pf-429242) or active (Ceapin-A1, Ceapin-A3) or inactive (Ceapin-A5) Ceapin analogs for one hour prior to harvesting lysates for Western Blot analysis of 3xFLAG-ATF6α. For both A and B inhibitor concentrations were 0.5, 5, 15, 25 μM respectively. GAPDH is shown as a loading control. Black arrowheads – 3xFLAG-ATF6α, white arrowheads – 3xFLAG-ATF6α-N. White lines in A indicate where intervening lane has been removed.