Figure 5.

CReP RNAi activates CHOP::GFP, an ISR target gene. (A) FACS^®-derived fluorescent profiles of nontransfected, CReP RNAi and CD2 RNAi transfected parental CHOP::GFP cells or CHOP::GFP cells stably overexpressing GADD34. The profile of nontransfected cells is shown in black. The light and dark tracings were obtained 16 and 28 h after RNAi transfection. (B) Immunoblots of eIF2α phosphorylated on serine 51, detected with an epitope-specific primary antiserum, eIF2α (P), total eIF2α(T), and PERK, which detects both the unphosphorylated, inactive form of the kinase (PERK°) and activated, phosphorylated form PERK(P) on lysates prepared from mouse ES cells with a CReP knockdown elicited by stable transduction of a CReP shRNA expressing plasmid or control scrambled shRNA plasmid. The cells were treated for 30 min with 1 mM dithiothreitol (DTT) and placed in DTT-free media for the indicated period of time (wash). (C) Immunoblot of endogenous CReP and PP1c in lysates of parental ES cells, CReP shRNA cells, and cells stably transfected with the control scrambled shRNA plasmid. The abundantly expressed TLS protein, detected with a specific antiserum, served as a loading control in this gel.