Knockdown of NIK expression by RNA interference inhibits both NF-κB pathways. (A) Immunoblot of NIK protein levels after infection of NIK shRNA in MMCLs that have cIAP1/2 deletion. (B) Knockdown of NIK affects NF-κB signaling via classical and alternative pathways. Nuclear extracts were prepared, and expression of the proteins indicated at right was analyzed on Western blots. NF-κB mutations in different MMCLs are indicated. (C) Knockdown of NIK inhibits nuclear NF-κB DNA binding. DNA-binding activity of the indicated NF-κB subunits was quantified by enzyme-linked immunosorbent assay. (D) Inhibition of NF-κB target gene expression after knockdown of NIK in MM cells. The −ΔNF-κB(3.1) = decreased expression for average of 3 target genes (TNFAIP3, NFKB2, and IL2RG) in MM cells treated with NIK shRNA versus empty vector. Data are mean ± SE.