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. 2021 Mar 25;95(8):e01990-20. doi: 10.1128/JVI.01990-20

FIG 1.

FIG 1

M50-mediated IRE1 degradation is blocked by proteasome inhibition. (A) MEFs were transfected with plasmids expressing Myc-tagged IRE1 and FLAG-tagged full-length or truncated (1–276) M50 or an empty vector (EV). Cells were treated with 25 μM MG-132 or DMSO for the last 5 1/2 h before harvesting of lysates at 48 h posttransfection. (B) MEFs were infected with MCMV-GFP (multiplicity of infection [MOI] = 3). Cells were treated with 30 μM MG-132 or DMSO for the last 6 h before harvesting. Cell lysates were analyzed by immunoblotting. M57 was detected as an infection control. (C) MEFs were transfected with a plasmid expressing HA-tagged K48-only ubiquitin and plasmids as described above for panel A. Cells were treated with 30 μM MG-132 or DMSO for the last 6 h before harvesting of lysates at 48 h posttransfection. IRE1 was immunoprecipitated (IP) with an anti-Myc antibody. Ubiquitylated proteins in whole-cell lysates (WCL) and in the immunoprecipitated samples were detected by immunoblotting with an anti-HA antibody.