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. 2017 Jan 31;91(4):e01138-16. doi: 10.1128/JVI.01138-16

FIG 1.

FIG 1

RNF31 interacts with LMP1 and IRF7. (A to C) 293T cells in 60-mm dishes were transfected with 1 μg Flag-RNF31 or Myc-RNA31; 0.5 μg HA-LMP1, Myc-LMP1, or Flag-LMP1 and its mutants; and 0.5 μg HA-IRF7 using Effectene transfection reagent. Cells were collected 48 h after transfection, and cell lysates in NP-40 lysis buffer were subjected to immunoprecipitation with anti-Flag M2 (Sigma) and protein A/G beads (Santa Cruz). After extensive washes, the beads were subjected to immunoblotting (IB) with anti-HA HA7 (Roche). (D and E) Cell lysates were prepared with NP-40 lysis buffer from IB4, JiJoye, and SavIII cells and were then subjected to preclearing with rabbit (D) or mouse (E) serum before immunoprecipitation with rabbit anti-RNF31 (Abcam) (D) or mouse anti-LMP1 (Dako) (E) or corresponding serum as a control. After extensive washes, the beads were subjected to immunoblotting with the indicated antibodies. Inputs were 5% of total cell lysates.