Figure 2.

LMP1 interacts with Src. (a) LMP1 interacts with Src in vitro. 293T cells in 60-mm dishes were transfected with 1 μg HA-LMP1 (or Flag-c-Src) and 1 μg Flag-IRF4 expression plasmids. Cells were collected 48 h post-transfection and then subjected to immunoprecipitation with the Flag antibody clone M2. Beads were washed extensively and then subjected to immunoblotting with the HA antibody clone HA-7. Inputs (5%) were subjected to immunoblotting with the indicated antibodies. (b) Endogenous LMP1 and Src interact in EBV latency. Cell lysates (approximately 1 mg of total proteins in each sample) prepared from JiJoye, IB4 and SavIII cells were subjected to immunoprecipitation with the c-Src antibody clone N16 (or rabbit serum as control). Beads were washed extensively and probed with LMP1 and Src antibodies. Inputs (5%) were also probed with these antibodies. (c) A diagram showing the LMP1 deletion mutant LMP1Δ(12–20). (d) The LMP1 SH3-binding region is not required for Src interaction. 293T cells in 60-mm dishes were transfected with 1 μg LMP1 or LMP1Δ(12–20) and 1 μg Flag-c-Src expression plasmids. Cells were collected 48 h post-transfection and then subjected to immunoprecipitation with the LMP1 antibody clone CS1-4. Beads were washed extensively and then subjected to immunoblotting with the Flag antibody clone M2 and the LMP1 antibody. Inputs (5% of total lysates) were subjected to immunoblotting with indicated antibodies. (e) The LMP1 SH3-binding region is not required for IRF4 activation. The 293 cells in 24-well plates were transfected with 50 ng IRF4 and 50 ng LMP1 (or LMP1Δ(12–20)) expression plasmids, 40 ng pGL3/IFNβ-Luc and 10 ng Renilla. Dual luciferase assay was performed. Results are the averages ± s.e. of duplicates. Representative results from at least three independent experiments are shown. The ability of the vector control to activate the promoter construct was set to 1.