NIK protein is overexpressed in established ATL and Hodgkin Reed-Sternberg cells. (A) Steady-state levels of NIK expression in the ATL and H-RS cell lines were revealed by immunoprecipitation-coupled immunoblotting. Approximately 2 × 107 cells were lysed with buffer A. After preclearing, immunoprecipitation was performed at 4°C, using anti-NIK antibody (NIK) or its isotype IgG (IgG). After 3 washes with TNT buffer, immune complexes were analyzed by immunoblotting with anti-NIK antibody. (B) 293T cells were transfected with pMRX-HA-iresPuro or pMRX-HA-NIKiresPuro for 24 hours. Whole-cell lysates were used as negative and positive controls. ED40515(−) cells were pretreated with (+) or without (−) MG132 (20 μM) for 3 hours, lysed with RIPA buffer, and subjected to immunoblotting with anti-NIK or anti-α-tubulin antibodies. Immunoprecipitation-coupled immunoblotting was performed as in panel A. (C) Top panels: control T-cell lines (CEM and Jurkat), leukemic cell lines derived from ATL patients that do not express Tax (ED40515(−), ATL43-Tb(−), and TL-Om1, a control B-cell line (RG69), and H-RS cell lines (HDLM-2 and L540) were pretreated with (+) or without (−) MG132 (20 μM) for 3 hours, and 30 μg of the whole-cell extracts were subjected to Western blot analysis with the antibodies to the indicated proteins. Bottom panels: Whole-cell extracts from the indicated cell lines were analyzed by Western blotting with the antibodies to the indicated proteins. (D) Total RNA was extracted from the indicated cell lines and subjected to real-time RT-PCR to quantify the NIK mRNA levels. The NIK mRNA levels were normalized to 18S RNA. The relative NIK mRNA levels shown represent the fold increases in mRNA abundance, relative to that of the CEM cells (arbitrarily set at 1). (E) Cells were cultured in the presence of actinomycin D (5 μg/mL) for the times indicated, and then total RNA was isolated and subjected to quantitative RT-PCR as in panel D. Data are expressed as mean plus or minus SD of 3 independent experiments. The relative amounts of NIK mRNA shown represent the percentages in mRNA abundance, relative to that of each cell line before the addition of actinomycin D (arbitrarily set at 100%). IB indicates immunoblotting; IP, immunoprecipitation.
