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. Author manuscript; available in PMC: 2022 Jun 16.
Published in final edited form as: J Immunol. 2020 Aug 3;205(6):1564–1579. doi: 10.4049/jimmunol.1901489

FIGURE 5.

FIGURE 5.

MERS-CoV ORF8b interacts with HSPs as determined by mass spectrometry. (A) Expression constructs of FLAG-tagged RLR pathway effectors were individually cotransfected with ORF8b-V5 plasmid into HEK293 cells as indicated. Transfected cells were harvested 40 h posttransfection and subjected to immunoprecipitation (IP) with anti-FLAG Ab. The total cell lysate and Ab-bound fraction were resolved by SDS-PAGE and analyzed by Western blotting with anti-V5 and anti-FLAG Abs. β-Actin was also detected as an internal loading control. (B) HEK293 cells were transfected with either ORF8b-V5 plasmid or empty vector. Transfected cells were harvested for immunoprecipitation with anti-V5 Ab 40 h posttransfection. Immunoprecipitates were then resolved in denaturing SDS-PAGE gel followed by silver staining for band visualization. Unique major bands (indicated by arrows a, b, and c) in ORF8b-V5 sample were cut and subjected to mass spectrometric analysis. HSPs identified to be interacting with ORF8b were listed in the table. (C) A table listing the proteins identified in bands a, b, and c by mass spectrometry.