Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, promotes Fn14 self-association on the cell surface.

<p>(<b>A</b>) HEK293 cells were transfected with vector or each of the Fn14-myc expression plasmids. At 24 hr post-transfection the cells were stained with FITC-labeled anti-myc antibody and analyzed by flow cytometry. The markers indicate the percentage of myc-positive cells. The values shown are from one representative experiment of two independent experiments. (<b>B</b>) HEK293/NF-κB-luc/Fn14-HA cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were either left untreated or treated with the cross-linking reagent BS3 as indicated. 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). Two different x-ray film exposures are shown for the BS3-treated samples.</p

Transient Fn14-FL or Fn14-ΔEC overexpression, but not Fn14-ΔCT overexpression, can activate the NF-κB signaling pathway.

<p>(<b>A</b>) 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>B</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). (<b>C</b>) 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.</p

The Fn14-FL and Fn14-ΔEC proteins, but not the Fn14-ΔCT protein, have slower electrophoretic mobility when analyzed under non-reducing conditions due to intermolecular disulfide bond formation during cell lysis.

<p>(<b>A</b>) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested, lysed, and equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions. Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (<b>B</b>) HEK293 cells were transfected with vector (V) or the Fn14-FL-myc expression plasmid and 24 hr later the cells were harvested. Cells were lysed and an aliquot of the Fn14-FL lysate was incubated with iodoacetamide (IA). Equal amounts of protein were subjected to SDS-PAGE under non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-GAPDH antibody. The positions of molecular size markers are shown on the left (in kDa).</p

Fn14-FL dimerization during cell lysis can be abrogated by mutagenesis of cysteine residue 122.

<p>(<b>A</b>) The human Fn14-WT (FL), Fn14-ΔCT, Fn14-C104S, and Fn14-C122S cytoplasmic tail amino acid sequences are shown with cysteine residues in red and the serine residue substitutions in blue. (<b>B</b>) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids and 24 hr later the cells were harvested and lysed. Equal amounts of protein were subjected to SDS-PAGE under either reducing or non-reducing conditions and Western blot analysis was performed using either an anti-myc or anti-tubulin antibody. The positions of molecular size markers are shown on the left (in kDa). (<b>C</b>) Schematic representation of predicted Fn14 cysteine residue status prior to and after cell harvesting and lysis. The Fn14 extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated and the cysteine residue distribution within the Fn14-FL protein is shown. Disulfide bonds are indicated with brackets. Non-covalent Fn14 association mediated by the most distal 18-aa residues of the Fn14 CT is illustrated with a double-headed arrow.</p

Cloning of a human Fn14 mRNA predicted to encode an Fn14 protein missing most of the extracellular domain.

<p>(<b>A</b>) Schematic representation of human Fn14 gene organization (via UCSC Genome Browser). The four Fn14 exons are numbered and boxed and the intron sizes are indicated in nucleotides (nt) at the top. The Fn14 amino acid (aa) numbers (1–129) and mature mRNA nucleotide (nt) numbers (1–1033) are provided above or below each exon, respectively. The positions of the two oligonucleotide primers used for RT-PCR analysis are indicated with arrows. (<b>B</b>) RNA was isolated from MDA-MB-231 and U87 cells and RT-PCR was performed using an exon 1 sense primer and an exon 4 antisense primer. PCR was also performed with this primer pair in the absence of cDNA (NT, no template). Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The positions of DNA size markers (M) are shown on the left (in base pairs). The two PCR products that were isolated and sequenced are indicated on the right as a and b. (<b>C</b>) Predicted amino acid sequence of PCR amplification products a and b. The last six amino acids of the signal peptide are indicated with an arrow, the Fn14 extracellular domain is bracketed with asterisks and the six cysteine residues found in this domain are in red. The Fn14 transmembrane domain is overlined and the cytoplasmic tail is underlined. (<b>D</b>) The Fn14 mRNA translation initiation codon and selected codons surrounding Fn14 introns 1 and 2 are shown for the Fn14 full-length (Fn14-FL) and Fn14 extracellular domain deletion (Fn14-ΔEC) mRNAs. The predicted Fn14-FL and Fn14-ΔEC amino acid sequence is shown below the nucleotide sequence. Abbreviation: I, intron. (<b>E</b>) Schematic representation of the Fn14-FL and Fn14-ΔEC proteins showing structural domains in relation to their exon coding regions. Amino acid numbers corresponding to the beginning of each protein domain and the C-terminal amino acid are indicated at the top of each diagram. The region of the extracellular domain that is missing in Fn14-ΔEC is shown in black. Abbreviations: SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CYT, cytoplasmic tail.</p

TWEAK cannot bind to the Fn14-ΔEC protein.

<p>(<b>A</b>) Schematic representation of the expression constructs encoding the Fn14-FL or Fn14-ΔEC protein. The Fn14 signal peptide (SP) region, extracellular (EC) domain, transmembrane (TM) domain, and cytoplasmic tail (CT) are indicated, the size of each EC domain is provided, and the positions of the signal peptide cleavage site (scissors) and the myc epitope tag are shown for both constructs. (<b>B</b>) HEK293 cells were transfected with vector or the indicated Fn14-myc expression plasmids. Cells were harvested 24 hr later, lysed, and equal amounts of protein were subjected to ligand blot analysis and Western blot analysis using either the anti-Fn14 #3600 antibody or an anti-tubulin antibody. The positions of molecular size markers are shown on the right (in kDa).</p