Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response
Figure 2
(A) NIH-3T3 cells stably expressing TEV-HA-tagged IRE1 were mock infected or infected with MCMV at an MOI of 5. Whole cell lysates (WCL) were applied to an anti-HA sepharose matrix. IRE1 and interacting proteins were eluted by TEV protease digestion, separated by SDS-PAGE, and silver stained. Specific bands (arrow heads) were excised and analyzed by protein mass spectrometry. (B) 293A cells were cotransfected with expression plasmids for IRE1-HA and Flag-tagged M50, m144, or Calnexin (CNX), respectively. IRE1 was subjected to immunoprecipitation (IP) with an anti-HA antibody. IRE1 and coexpressed proteins were detected by immunoblot in IP samples and WCL using anti-HA and anti-Flag antibodies, respectively. (C) 293A cells were cotransfected with expression plasmids for IRE1-HA and Flag-tagged M50 or m144, respectively. M50 and m144 were precipitated with an anti-Flag antibody. IRE1 and coexpressed proteins were detected in IP samples and WCL as described above. (D) 10.1 fibroblasts transduced with a retroviral vector expressing myc-tagged IRE1 were infected with an MCMV expressing HA-tagged M50 or m41 at an MOI of 4. At the indicated time points IRE1 was immunoprecipitated with an anti-myc antibody, and HA-tagged proteins were detected by immunoblot. (E) NIH-3T3 cells were infected with the same viruses as in D. After 24 h, M50 and m41 proteins were immunoprecipitated with an anti-HA antibody. IRE1 was detected in IP samples and WCL using an IRE1-specific antibody.