CD44ICD activates NF-κB. (A) Raw 264.7 cells were cotransfected with pcDNA3-CD44ICD and NF-κB, AP-1 or NFAT luciferase reporter constructs, grown for 2 days, and then either assayed for luciferase activity (^*P < .05 and ^**P < .001 versus 0 μg CD44ICD; SD; n = 3) or subjected to Western blot analysis using anti-myc antibody. RANKL was used as a positive control. (B) Raw 264.7 cells were cotransfected with pcDNA3-CD44FL or pcDNA3-CD44ΔE and NF-κB luciferase reporter construct, grown for 2 days, and either assayed for luciferase activity (^**P < .001 versus 0 μg CD44FL or CD44ΔE; SD; n = 3) or subjected to Western blot analysis using anti-myc antibody. RANKL was used as a positive control. (C) Raw 264.7 cells were transduced or not with MgR1-CD44ICD and stimulated or not for 30 minutes with LPS (100 μg/mL) or RANKL (100 ng/mL). Nuclear proteins were extracted and incubated for 20 minutes with radiolabeled NF-κB consensus oligonucleotides. Some cells were preincubated with NEMO-binding peptide (NBD; wt; 50 μM) or NBD mutant peptide (mut; 50 μM) for 1 hour at room temperature. The samples were resolved by electrophoresis. (D) Raw 264.7 cells were transduced with MgR1-CD44ICD and stimulated or not with RANKL (100 ng/mL) and then nuclear extracts were analyzed as in panel B, except that some nuclear extracts were preincubated with an antibody directed against p65, myc, or with both p65 and myc. (E) Raw 264.7 cells were transduced with MgR1 empty or encoding CD44ICD and stimulated with RANKL (100 ng/mL) for the indicated times. Total cell extracts were analyzed by Western blotting using antibodies directed against I-κB, phosphorylated I-κB (P-I-κB), and GAPDH.