![Characterization of NF-κB and IKK activity in ALCL cells. (A) EMSA analysis of unstimulated cells. Nuclear extracts of unstimulated HRS and ALCL cell lines were analyzed for NF-κB DNA binding activity by EMSA. Positions of NF-κB and (p50)2 complexes are indicated. n.s. indicates nonspecific. (B) Induction of NF-κB p50-p65 activity in K299 ALCL cells. Nuclear extracts of unstimulated or TNF-α–treated K299 cells were analyzed with or without preincubation with the indicated antibodies for supershift (ss) analysis by EMSA for NF-κB DNA binding activity. Positions of NF-κB and (p50)2 complexes are indicated. n.s. indicates nonspecific. (C, D) Analysis of IKK activity in unstimulated or TNF-α–treated cell lines, as indicated. (C) IKK activity in unstimulated cells was determined by an in vitro kinase assay (KA). IKKα was immunoprecipitated using a monoclonal antibody against IKKα and protein A–sepharose. The precipitates were incubated with GST-IκBα (aa 1-53) and γ-[32P]ATP at 37°C and subsequently analyzed by SDS-PAGE and autoradiography. As control, IKKα expression was analyzed by Western blot. (D) Prior to IKK analysis, cells were treated for 15 minutes with TNF-α. Analysis of the IKK activity was as described in panel C. Note that in panel D the IKK activity between the different cell lines is not comparable.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/106/13/10.1182_blood-2004-09-3620/2/zh80240588080001.jpeg?Expires=1721367432&Signature=0veZNAcUAbMbvD-08k5YqBUHLrMuGMZb~ljlQN9wQNwIlbd1hj9ewGvCEGlmV95DilxMZ6FUbA7ZG~hhHTdzGiJGqrAqpvHg27HUmxxp0IOaFN2dN8kIHwjnHedowqFf-hWN~JZv-9hX7FnDJY0xmIRM6RNDAj908k0y-R1EpcsbmZnQZjJj-wdTDvD4sLxOIE4sp3TBDd71TOktK4jFGQ8C8Hn6TrfAwkciplCmnt-2g80E9qGlDT4c1O3cDl9~ETNmFkxYWYQTUoDA7tC28RfNkwpFca9Sc4PWQHVCQJYm00Ep16~o99KH4vAD4zV8HTyo5OOsnxJ3HuTNnrZ5HQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Characterization of NF-κB and IKK activity in ALCL cells. (A) EMSA analysis of unstimulated cells. Nuclear extracts of unstimulated HRS and ALCL cell lines were analyzed for NF-κB DNA binding activity by EMSA. Positions of NF-κB and (p50)2 complexes are indicated. n.s. indicates nonspecific. (B) Induction of NF-κB p50-p65 activity in K299 ALCL cells. Nuclear extracts of unstimulated or TNF-α–treated K299 cells were analyzed with or without preincubation with the indicated antibodies for supershift (ss) analysis by EMSA for NF-κB DNA binding activity. Positions of NF-κB and (p50)2 complexes are indicated. n.s. indicates nonspecific. (C, D) Analysis of IKK activity in unstimulated or TNF-α–treated cell lines, as indicated. (C) IKK activity in unstimulated cells was determined by an in vitro kinase assay (KA). IKKα was immunoprecipitated using a monoclonal antibody against IKKα and protein A–sepharose. The precipitates were incubated with GST-IκBα (aa 1-53) and γ-[32P]ATP at 37°C and subsequently analyzed by SDS-PAGE and autoradiography. As control, IKKα expression was analyzed by Western blot. (D) Prior to IKK analysis, cells were treated for 15 minutes with TNF-α. Analysis of the IKK activity was as described in panel C. Note that in panel D the IKK activity between the different cell lines is not comparable.
