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

FIG 4.

FIG 4

HAdV infection promotes RNF4-Daxx interaction and Daxx ubiquitin PTM. (A) H1299 cells were infected with wt virus (H5pg4100) at a multiplicity of 50 FFU per cell (left), cotransfected with 2 μg of HA-Daxx prior to infection, and harvested 16 and 36 h postinfection, and then 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), Daxx rabbit pAb 07-471 (Upstate), and mouse MAb AC-15 (α-β-actin) as a loading control. Note that light chains (IgG) are detected at 20 kDa. Molecular sizes, in kDa, are indicated on the left, and relevant proteins are on the right. (B) H1299 cells were stably transfected with 5 μg of pRNF4-Flag and pE1B-55K. Cells were treated with 25 mM NEM and 10 μM Mg132 and incubated for an additional 4 h. At 28 h posttransfection, cell pellets were resuspended in 1% SDS lysis buffer and cleared by centrifugation. Modification of Daxx was analyzed by immunoblotting after SDS-PAGE. Input levels of total cell lysates and immunoprecipitated Daxx were detected using Daxx rabbit pAb 07-471 (Upstate), MAb P4D1 (α-Ubiquitin), RNF4 mouse pAB A01 (Abnova), and MAb 2A6 (α-E1B-55K). Molecular sizes, in kDa, are indicated on the left, and relevant proteins are on the right. (C) H1299 cells were stably transfected with 10 μg pUbiquitin-His and 5 μg each of pDaxx-HA, pRNF4-Flag, and either pE1B-55K or pE1B-55K-SCM. Cells were treated with 25 mM NEM, and total cell lysates were prepared with guanidinium chloride buffer 28 h posttransfection and subjected to Ni-NTA purification of ubiquitin-His-conjugated proteins. Proteins were separated by SDS-PAGE and subjected to immunoblotting. Input levels of total cell lysates and Ni-NTA-purified proteins were detected using Daxx rabbit pAb 07-471 (Upstate), MAb 6×His (α-His), MAb AC-15 (α-β-actin), and MAb 2A6 (α-E1B-55K). Molecular sizes, in kDa, are indicated on the left, and relevant proteins are on the right.