Left panel, TMK-1 cells were transfected with control siRNA or siRNA targeting ADAM17, ADAM10 or their combination. At 48 h after transfection, medium was changed to RPMI containing 0.1% BSA. CM was prepared after the cells were cultured for additional 48 h, and TNF-α in CM was quantified by ELISA. Right panel, NF-κB p65 DNA-binding activity was measured at 72 h after each siRNA was introduced into TMK-1 cells.
At 72 h after TMK-1 cells were transfected with each siRNA, qRT-PCR was performed for the indicated genes.
qRT-PCR analyses for cyclin D1, c-Myc and Bcl-2 mRNA levels in the stable TMK-1 clones.
A subconfluent monolayer of TMK-1 cells was treated with 2 µg/ml control IgG, anti-TNF-α neutralizing antibody, or anti-IL-6 neutralizing antibody for 24 h. Then, mRNA levels were measured by qRT-PCR.
mRNA levels of the indicated genes in TMK-1 cells were quantified by RT-PCR after siRNA-mediated knockdown of ADAM17, ADAM10 or both genes for 72 h.