RANKL, a weak NF-κB activator, does not protect primary BMDM from MTX-induced apoptosis. A. RANKL (100 to 400 ng/mL) was introduced to BMDM 6 hours before MTX (10 mM) treatment. Caspase-3 activity was measured 24 hours later. Results are mean ± SEM from at least six independent experiments. B. and C. Compared to RANKL (200 ng/mL), TNF-a (10 ng/mL) treatment causes greater activation of NF-kB in BMDM. Cells transiently expressing the pNF-kB-TA-Luc reporter were stimulated with RANKL or TNF-a for six hours (B). Shown are mean ± SEM of normalised data from three independent experiments. C. Cells were M-CSF-starved overnight before stimulation with RANKL or TNF-a for 10 minutes and 30 minutes. Whole-cell lysates were subjected to western blot analysis for phosphorylated IkBa, total IkBa and b-actin. Results are representative of three independent experiments. * = P < 0.05; ** = P < 0.01.