LMP1 signaling complexes.

LMP1 associates with endogenous TAK1, TAB2 and TRAF6.
(A) 293 cells were transfected with expression vectors for various versions of Flag-LMP1. Cell lysates were immunoprecipitated with anti-Flag, and co-precipitated endogenous proteins were analyzed by immunoblotting. The precipitated of Flag-LMP1 is also shown. WCE: whole cell extracts; HC, immunoglobulin heavy chain.
(B) 293 cells were transfected with expression vectors for Flag-LMP1 WT or 1-186 mutant. Cell lysates were immunoprecipitated with anti-TAK1, and co-precipitated Flag-LMP1 was analyzed by immunoblotting. The precipitated TAK1 and TAB2 are also shown. WCE: whole cell extracts. The asterisk indicates that a partially degraded Flag-LMP1 is also coprecipitated with TAK1 and TAB2.

LMP1 associates with TAK1, TAB2 and TRAF6.
(A) Schematic drawing of wild type LMP1 and mutants.
(B-D) 293 cells were transfected with expression vectors for various versions of Flag-LMP1 or Flag-Transferrin Receptor (TfR) together with HA-TAK1 (B), HA-TAB2 (C) or HA-TRAF6 (D). Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE, whole cell extracts; HC, immunoglobulin heavy chain; LC, immunoglobulin light chain.

Effect of TAK1 inhibitor.
(A) 293 cells were transfected with empty vector or expression vectors for Flag-LMP1. 5Z-7-oxozeaenol was added to the culture medium every 12 hr after transfection and some cells were treated with 10 ng/ml TNF for 30 min prior to harvest. The activated form of JNK was detected with phospho-JNK antibody (P-JNK).
(B) 293 cells were transfected with NF-κB-dependent luciferase reporter and pAct-β-galactosidase plasmids, together with expression vectors for NIK or Flag-LMP1. 5Z-7-oxozeaenol was added to the culture medium at the concentrations indicated every 12 hr after transfection. Luciferase activity was determined and normalized to the levels of β-galactosidase activity. Stimulation relative to control transfection with empty vector is shown.

Effect of LMP1 mutations on activation of JNK and NF-κB.
(A) 293 cells were transfected with expression vectors for various versions of Flag-LMP1. Increasing amount of Flag-LMP1 1-186 vectors were used. The activated form of JNK was detected with phospho-JNK antibody (P-JNK).
(B) 293 cells were transfected with expression vectors for various versions of Flag-LMP1. The amounts of plasmids were same as (A). Cell lysates were subjected to electrophoretic mobility shift assay (EMSA) using NF-κB DNA probe (top panel). NF-κB binding was confirmed by supershift with anti-p65. Amounts of p65 in the cell lysates and expression levels of LMP1 are also shown. WCE: whole cell extracts.
(C) 293 cells were transfected with an NF-κB-dependent luciferase reporter and pAct-β-galactosidase plasmids, together with expression vectors for various versions of Flag-LMP1 or Flag-TfR. Increasing amount of Flag-LMP1 1-186 vectors were used. Luciferase activity was determined and normalized to the levels of β-galactosidase activity. Stimulation relative to control transfection with empty vector is shown.
(D) Left panels: 293 cells were transfected with expression vectors for various versions of Flag-LMP1 or Flag-Transferrin Receptor (TfR) together with HA-IKKγ. Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE: whole cell extracts. Right panels: 293 cells were transfected with expression vectors for T7-TAK1 together with HA-IKKγ in the absence or presence of Flag-LMP1 WT or 1-186 mutant. Cell lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). WCE: whole cell extracts.

TAK1 is essential for LMP1-induced JNK activation.
(A) Schematic drawing of TAK1δ
(B) TAK1 +/+, TAK1 Δ/Δ, and T7-TAK1-expressing TAK1 Δ/Δ MEFs were transfected with expression vectors for HA-JNK and Flag-LMP1. Some cells were treated with anisomycin for 30 min prior to harvest. HA-JNK was immunoprecipitated and the activated form of JNK was detected with phospho-JNK antibody (P-JNK). WCE: whole cell extracts.
(C) TAK1 +/+ and TAK1 Δ/Δ MEFs were transfected with expression vectors for Flag-LMP1. Cell lysates were subjected to electrophoretic mobility shift assay (EMSA) using an NF-κB DNA probe. NF-κB binding was confirmed by supershift with anti-p65. Amounts of p65 in the cell lysates and expression levels of LMP1 are shown. WCE: whole cell extracts.
(D) TAK1 +/+ and TAK1 Δ/Δ MEFs were transfected with NF-κB-dependent luciferase reporter and pEF1α Renilla luciferase plasmids together with expression vector for Flag-LMP1. Luciferase activity was determined and normalized to the levels of Renilla luciferase activity. Stimulation relative to control transfection with empty vector is shown.
(E) Left panels: Proteins from HeLaS3 control or TAK1 siRNA cells were immunoblotted with anti-TAK1 (upper panel) and anti-β-actin (lower panel). Right panels: HeLaS3 control or TAK1 siRNA cells were transfected with expression vectors for HA-JNK and Flag-LMP1. Some cells were treated with anisomycin for 30 min prior to harvest. HA-JNK was immunoprecipitated and the activated form of JNK was detected with phospho-JNK antibody (P-JNK). WCE: whole cell extracts.
(F) HeLaS3 control or TAK1 siRNA cells were transfected with AP-1- or NF-κB-dependent luciferase reporter and pEF1α Renilla luciferase plasmids together with expression vector for Flag-LMP1. Luciferase activity was determined and normalized to the levels of Renilla luciferase activity. Stimulation relative to control transfection with empty vector is shown.