American Society of Hematology

Figure 5

$Figure 5. NPI-0052 inhibits TNF-dependent NF-κB activation and IκBα degradation. (A) Cells were preincubated with 50 nM NPI-0052 for 4 hours, treated with 0.1 nM TNF for the indicated times, and then subjected to EMSA and Western blot analysis to evaluate NF-κB activation. (B) Cells were incubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed with anti-IκBα and anti-phospho-specific anti-IκBα. (C) Cells were preincubated with 50 nM NPI-0052 for 4 hours, incubated with 50 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 minutes, and then treated with 0.1 nM TNF for 15 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using phospho-specific anti-IκBα antibody. The same membrane was reblotted with anti-IκBα antibody. (D) Cells were preincubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for 15 minutes. Cytoplasmic extracts were immunoprecipitated with antibody against IκBα then subjected to Western blot analysis using monoclonal anti-ubiquitin antibody. (E) Cells were preincubated with 50 nM NPI-0052 for 4 hours and then treated with 1 nM TNF for the indicated times. Whole-cell extracts (1 mg/mL) were immunoprecipitated with antibody against IKK-α and analyzed using an immunocomplex kinase assay. Data are for a representative experiment of 3 independent ones showing similar results.$

NPI-0052 inhibits TNF-dependent NF-κB activation and IκBα degradation. (A) Cells were preincubated with 50 nM NPI-0052 for 4 hours, treated with 0.1 nM TNF for the indicated times, and then subjected to EMSA and Western blot analysis to evaluate NF-κB activation. (B) Cells were incubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed with anti-IκBα and anti-phospho-specific anti-IκBα. (C) Cells were preincubated with 50 nM NPI-0052 for 4 hours, incubated with 50 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 minutes, and then treated with 0.1 nM TNF for 15 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using phospho-specific anti-IκBα antibody. The same membrane was reblotted with anti-IκBα antibody. (D) Cells were preincubated with 50 nM NPI-0052 for 4 hours and then treated with 0.1 nM TNF for 15 minutes. Cytoplasmic extracts were immunoprecipitated with antibody against IκBα then subjected to Western blot analysis using monoclonal anti-ubiquitin antibody. (E) Cells were preincubated with 50 nM NPI-0052 for 4 hours and then treated with 1 nM TNF for the indicated times. Whole-cell extracts (1 mg/mL) were immunoprecipitated with antibody against IKK-α and analyzed using an immunocomplex kinase assay. Data are for a representative experiment of 3 independent ones showing similar results.

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