TNF-α mRNA level in the indicated stable cells was analysed by qRT-PCR.
Left panel, TNF-α protein expression in the cell lysates was quantified by ELISA. Right panels, same lysates were subjected to Western blotting using anti-TNF-α antibody recognizing the extracellular domain of TNF-α.
FLAG-pro-TNF-α (or equal amount of the empty vector) was expressed in the NC or NRDc-KD #1 TMK-1 cells as indicated. At 36 h after transfection, the cells were lysed and aliquots were probed with the indicated antibodies. tm-, transmembrane precursor; c-, N-terminal cytosolic remnant.
NF-κB transcriptional activity was analysed by luciferase assay in the NC or NRDc-KD #1 TMK-1 cells transfected with pNF-κB-Luc and pRL-TK reporter genes.
Nuclear extracts were prepared from the indicated cells, and DNA-binding activity of NF-κB p65 in each nuclear extract was measured.
The NRDc-KD #1 cells were stimulated with the increasing doses of recombinant TNF-α protein for 1 h. Then NF-κB DNA-binding activity was analysed.
TMK-1 cells were cultured in the presence of 2 µg/ml control IgG or anti-TNF-α neutralizing antibody for 24 h, and qRT-PCR was performed for the indicated genes.
IL-6 protein secretion into CM was quantified by ELISA after TMK-1 cells were treated with 2 µg/ml control IgG or anti-TNF-α neutralizing antibody for 48 h.
ELISA quantified the IL-6 concentration in CM from the NRDc-KD #1 cells incubated with the indicated concentrations of recombinant TNF-α for 48 h.
Cell viability was analysed with the MTS assay after TMK-1 cells were incubated with the indicated antibodies for 24 h.
The NC or NRDc-KD #1 TMK-1 cells seeded in 96-well plates were incubated with the increasing doses of TNF-α or vehicle for 24 h, followed by analysis using the MTS assay.
