Lack of response to TNF- in cells from double- and triple-KO mice.

BMSCs were stimulated with LPS (1 μg/mL) or TNF-α (10 ng/mL) for 3 hours. Cells were then harvested; RNA was extracted; and 20 μg of total cellular RNA was separated on a 1.5% agarose gel and used for Northern blotting with a mouse GM-CSF cDNA probe. The blots were exposed to autoradiographic film for 6 days. WT indicates wild-type mice; KO indicates TTP-deficient mice; 2KO indicates mice deficient in both TNF-α receptors, but WT for TTP; 3KO indicates mice deficient in TTP and both TNF-α receptors. C, control; L, LPS (1 μg/mL); T, TNF-α (10 ng/mL). The arrows indicate the positions of the 2 GM-CSF mRNA species. Note the absence of response to TNF-α in the cells derived from double- and triple-KO mice. The same blots were hybridized with a rat GAPDH cDNA probe as a loading control. Exposure of the blots to autoradiographic film for GAPDH mRNA was 4 hours.