Figure 6
Figure 6. Depletion of NIK suppresses NF-κB-dependent transcription in ATL cells. (A) ED40515(−) and ATL-43Tb(−) cells were infected with lentiviral vectors expressing Renilla luciferase (Ctli) or NIK-specific shRNAs (NIKi-1 or NIKi-2). In parallel, ED40515(−) and ATL-43Tb(−) cells were infected with lentiviral vectors expressing Ctli or NIKi-1 shRNAs, and 24 hours later, these cells were super-infected with lentiviral vectors expressing Ctli or NIKi-2 shRNAs. Twenty-four hours after infection, cells were selected with puromycin for 2 days. Puromycin-resistant cells were then transfected with 2 μg of IgκCona-Luc and 2 μg EF1-LacZ. Luciferase (LUC) activity was determined 48 hours after transfection and normalized to β-gal activity. Relative luciferase activities, in comparison with control cells, 100 are shown. Data are expressed as mean plus or minus SD of 3 independent experiments. P values are versus control (Ctli). (B) Super-infected cells were treated with or without MG132 (20 μM) for 3 hours and subjected to SDS-PAGE and immunoblotting with anti-NIK (#4994), antiphosphorylated p100, or anti-α-tubulin antibodies. Whole-cell extracts (30 μg) from these cells were analyzed by SDS-PAGE and immunoblotting with antiphospho-IκBα, anti-IκBα, or anti-α-tubulin antibodies. Cytoplasmic extracts prepared from ED40515(−) cells infected or not with lentivirus were precleared and immunoprecipitation was performed, using anti-IKK1 monolconal antibody or its isotype IgG (IgG). After 3 washes with TNT buffer, immune complexes were treated or not with Shrimp Akaline Phosphatase (Takara Bio) and then subjected to SDS-PAGE and immunoblotting with antiphospho-IKK1/2, anti-IKK1, or anti-IKK2 antibodies. (C) A total of 5 μg of nuclear extracts prepared from lentivirus-infected cells shown in panel B were analyzed by EMSA, using oligonucleotides encoding the NF-κB–binding sequence or Oct-1–binding sequence as probes. (D) Nuclear extracts (5 μg) from lentivirus-infected cells shown in panel B were preincubated for 30 minutes with purified mouse IgG, anti-p50, anti-cRel antibody, preimmune (PI), anti-p50, anti-RelA or anti-RelB sera, and then subjected to EMSA with the NF-κB–specific probe. (E) Total RNAs from lentivirus-infected cells shown in panel B were examined by quantitative RT-PCR for VEGF, ICAM-1, and MMP-9 mRNA levels. Each mRNA level was normalized to 18S RNA. Relative mRNA levels, in comparison with control cells, 100 are shown. Data are expressed as mean plus or minus SD of 3 independent experiments. P values are versus control (Ctli + Ctli).

Depletion of NIK suppresses NF-κB-dependent transcription in ATL cells. (A) ED40515(−) and ATL-43Tb(−) cells were infected with lentiviral vectors expressing Renilla luciferase (Ctli) or NIK-specific shRNAs (NIKi-1 or NIKi-2). In parallel, ED40515(−) and ATL-43Tb(−) cells were infected with lentiviral vectors expressing Ctli or NIKi-1 shRNAs, and 24 hours later, these cells were super-infected with lentiviral vectors expressing Ctli or NIKi-2 shRNAs. Twenty-four hours after infection, cells were selected with puromycin for 2 days. Puromycin-resistant cells were then transfected with 2 μg of IgκCona-Luc and 2 μg EF1-LacZ. Luciferase (LUC) activity was determined 48 hours after transfection and normalized to β-gal activity. Relative luciferase activities, in comparison with control cells, 100 are shown. Data are expressed as mean plus or minus SD of 3 independent experiments. P values are versus control (Ctli). (B) Super-infected cells were treated with or without MG132 (20 μM) for 3 hours and subjected to SDS-PAGE and immunoblotting with anti-NIK (#4994), antiphosphorylated p100, or anti-α-tubulin antibodies. Whole-cell extracts (30 μg) from these cells were analyzed by SDS-PAGE and immunoblotting with antiphospho-IκBα, anti-IκBα, or anti-α-tubulin antibodies. Cytoplasmic extracts prepared from ED40515(−) cells infected or not with lentivirus were precleared and immunoprecipitation was performed, using anti-IKK1 monolconal antibody or its isotype IgG (IgG). After 3 washes with TNT buffer, immune complexes were treated or not with Shrimp Akaline Phosphatase (Takara Bio) and then subjected to SDS-PAGE and immunoblotting with antiphospho-IKK1/2, anti-IKK1, or anti-IKK2 antibodies. (C) A total of 5 μg of nuclear extracts prepared from lentivirus-infected cells shown in panel B were analyzed by EMSA, using oligonucleotides encoding the NF-κB–binding sequence or Oct-1–binding sequence as probes. (D) Nuclear extracts (5 μg) from lentivirus-infected cells shown in panel B were preincubated for 30 minutes with purified mouse IgG, anti-p50, anti-cRel antibody, preimmune (PI), anti-p50, anti-RelA or anti-RelB sera, and then subjected to EMSA with the NF-κB–specific probe. (E) Total RNAs from lentivirus-infected cells shown in panel B were examined by quantitative RT-PCR for VEGF, ICAM-1, and MMP-9 mRNA levels. Each mRNA level was normalized to 18S RNA. Relative mRNA levels, in comparison with control cells, 100 are shown. Data are expressed as mean plus or minus SD of 3 independent experiments. P values are versus control (Ctli + Ctli).

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