TWEAK-Independent Fn14 Self-Association and NF-κB Activation Is Mediated by the C-Terminal Region of the Fn14 Cytoplasmic Domain
Figure 3
Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.
(A) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔCT protein. The Fn14-FL construct is the same as shown in Fig. 2A but here the size of the cytoplasmic tail (CT) is indicated. The Fn14-ΔCT construct contains an Ig-κ chain signal peptide instead of the Fn14 signal peptide and an extra stretch of 9-aa residues that was introduced during the cloning procedure (denoted here as L for linker). The positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (B) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and grown for 24 hr. Cells were harvested, lysed, and equal amounts of protein were used for Western blot analysis using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (C) HEK293/NF-κB-luc cells were transfected in 6 well dishes with the indicated plasmids and grown for 24 hr. The cells were harvested, lysed, and NF-κB-luc reporter expression was assayed using a luminometer. Luciferase activity was normalized to the vector-transfected cells. The values shown are mean +/− SEM of triplicate wells from one representative experiment of three independent experiments. *P<0.01 versus vector using Student’s t test.