Figure 4
Determination of ISR kinase requirement for induction of ATF4 and CHOP by GSK-J4, and assessment of HRI modification. (A) Requirement of HRI for ATF4 and CHOP induction. MEFs lacking expression of all the four ISR kinases (4KO) and expressing one of the kinases by exogenous complementation (+ PERK, + PRK, + GCN2 or + HRI) were tested for their ability to induce ATF4 and CHOP by 30 µM GSK-J4 treatment for 0, 90 or 240 min. (B) Phosphorylation of eIF2α. MEFs expressing HRI (MEF-4KO + HRI) were treated with GSK-J4 and analyzed by western blotting. (C) HRI migration change in gel. Protein samples from MEF-4KO + HRI were treated with SDS-PAGE sample buffer with no reducing agent for 5 min at 40 °C. Upper and lower band area were dissected and shown below with horizontal red lines to compare migration. Note that GSK-J4-treated HRI bands migrate more slowly or were shifted up. (D) Treatment with phosphatase. Extracts from MEF-4KO + HRI treated with GSK-J4 for 90 min were further treated in vitro with CIAP for 10 or 30 min at 30 °C and analyzed by western blotting. E, Disruption of high molecular weight bands by a reducing reagent. Extracts from GSK-J4-treated MEF-4KO + HRI were treated with SDS PAGE sample buffer containing no (left panel) or 1% 2-mercaptoethanol (right panel) for 5 min at 40 °C.
