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. 2018 Jun 13;92(13):e00164-18. doi: 10.1128/JVI.00164-18

FIG 2.

FIG 2

E1B-55K interaction with RNF4 protein. (A) H1299 cells were cotransfected with 2 μg pFlag-RNF4-WT and 2 μg pE1B-55K. After 24 and 48 h posttransfection (h p.t.), cells were fixed with 4% PFA and labeled with α-Flag mouse MAb M2 (Sigma-Aldrich, Inc.), detected with Alexa 488 (α-Flag; green) and mouse MAb 2A6 (α-E1B-55K), and detected with Cy3 (α-E1B-55K; red)-conjugated secondary antibody. Mock cells are transfected but not infected. Nuclei are labeled with DAPI (4,6-diamidino-2-phenylindole). Representative α-Flag (green; Db, Df, Dj), α-E1B-55K (red; Dc, Dg, Dk), DAPI (blue; Da, De, Di) staining patterns and overlays of the single images (merge; Dd, Dh, Dl) are shown (magnification, ×7600). (B) H1299 cells were transfected with an empty vector control or a plasmid encoding E1B-55K and harvested 48 h posttransfection, or they were infected with wt virus (H5pg4100) at a multiplicity of 50 FFU per cell and harvested 24 h postinfection, and total cell extracts were prepared. Immunoprecipitation of endogenous RNF4 was performed using RNF4 mouse pAb A01 (Abnova), and proteins were separated by SDS-PAGE and subjected to immunoblotting. Input levels of total cell lysates and coprecipitated proteins were detected using mouse MAb 2A6 (α-E1B-55K), RNF4 mouse pAb A01 (Abnova), and mouse MAb AC-15 (α-β-actin) as a loading control. Note that heavy chains (IgH) are detected at 55 kDa. Molecular sizes in kDa are indicated on the left, and relevant proteins are on the right. (C) H1299 cells were cotransfected with 5 μg pFlag-RNF4-WT and 3 μg pE1B-55K-wt, 6 μg SCS (SUMO conjugation site K104R mutant), or 1.5 μg NES (nuclear export signal mutant) and harvested 48 h posttransfection, and total cell extracts were prepared. Immunoprecipitation of pFlag-RNF4 was performed using α-Flag mouse MAb M2 (Sigma-Aldrich, Inc.), and proteins were separated by SDS-PAGE and subjected to immunoblotting. Input levels of total cell lysates and coprecipitated proteins were detected using the Ab indicated in panel B. Molecular sizes, in kDa, are indicated on the left, and relevant proteins are on the right.