Nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) is dispensable for tumour necrosis factor (TNF) receptor but is essential for lymphotoxin beta-receptor (LTβR)-induced NF-κB activation in primary human skin fibroblasts. Primary human skin fibroblasts were infected with Adv.GFP, Adv.NIK-kd, Adv.IKK2-kd or Adv.IκBα (moi 50) and left for 24 hours to allow transgene expression. (a) Cells were stimulated with vehicle (control), with 25 ng/ml TNF-α or with 50 ng/ml LTα1β2 for 30 min prior to the preparation of cytosolic and nuclear extracts. Cytosolic IκBα levels were determined by western blotting (upper panel). Nuclear NF-κB DNA-binding activities were determined by electrophoretic mobility shift assay (EMSA) (lower panel). (b) Cells were stimulated with vehicle (control), with 25 ng/ml TNF-α or with 50 ng/ml LTα1β2 for 24 hours, and secreted IL-6 levels were determined by enzyme-linked immunosorbent assay (n = 3, ± SEM).