Figure 3.

MYXV differentially activates each arm of the UPR. MYXV-treated U266 cells were harvested 6 hours postinfection. (A) RNA extracted from U266 cells was subjected to qPCR. The resulting PCR product was then digested with Pst1, loaded onto an 8% DNA acrylamide gel, and stained with SYBR^® Gold. PCR products derived from XBP1 mRNA, which had undergone IRE1-mediated splicing, contain a Pst1-resistant band of higher molecular weight (arrow). (B) U266 lysate was subjected to treatment with 50 mM DTT prior to loading onto a Laemmli gel. Samples were then immunoblotted for ATF6. Full-length ATF6 (arrow) is lost following activation. (C) U266 lysate was separated on a Laemmli gel and then immunoblotted with antibodies against PERK. Phosphorylated PERK is observed as a high-molecular-weight band (arrow).

Abbreviations: ATF, activating transcription factor; BreA, brefeldin-A; DTT, dithiothreitol; IRE1, inositol-requiring protein-1; mRNA, messenger RNA; MYXV, myxoma virus; PCR, polymerase chain reaction; PERK, protein kinase RNA-like endoplasmic reticulum kinase; qPCR, quantitative real-time polymerase chain reaction; UPR, unfolded protein response.