American Society of Hematology

Figure 2.

$Figure 2. Effect of I3C on IκBα phosphorylation and degradation induced by TNF. (A) I3C inhibits TNF-induced activation of NF-κB. Jurkat cells were incubated with 50 μM I3C for 24 hours, treated with 0.1 nM TNF for indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of I3C on TNF-induced degradation of IκBα. Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκBα antibody. Anti-β-actin antibody was the loading control. (C) Effect of I3C on the phosphorylation of IκBα by TNF. Jurkat cells were incubated with of 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were fractionated and then blotted using phosphospecific anti-IκBα antibody. (D) Effect of I3C on TNF-induced ubiquitination of IκBα. Jurkat cells were pretreated with 50 μM I3C for 24 hours, then with 100 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 1 hour, and finally with 0.1 nM TNF for 15 minutes. Whole-cell extracts were prepared, fractionated, and examined by Western blot analysis using anti-IκBα antibody. (E) Effect of I3C on the activation of IKK by TNF. Jurkat cells were incubated with 50 μM I3C for 24 hours and then activated with 1 nM TNF for different times. Whole-cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by immunocomplex kinase assay. To examine the effect of I3C on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies.$

Effect of I3C on IκBα phosphorylation and degradation induced by TNF. (A) I3C inhibits TNF-induced activation of NF-κB. Jurkat cells were incubated with 50 μM I3C for 24 hours, treated with 0.1 nM TNF for indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of I3C on TNF-induced degradation of IκBα. Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκBα antibody. Anti-β-actin antibody was the loading control. (C) Effect of I3C on the phosphorylation of IκBα by TNF. Jurkat cells were incubated with of 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were fractionated and then blotted using phosphospecific anti-IκBα antibody. (D) Effect of I3C on TNF-induced ubiquitination of IκBα. Jurkat cells were pretreated with 50 μM I3C for 24 hours, then with 100 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 1 hour, and finally with 0.1 nM TNF for 15 minutes. Whole-cell extracts were prepared, fractionated, and examined by Western blot analysis using anti-IκBα antibody. (E) Effect of I3C on the activation of IKK by TNF. Jurkat cells were incubated with 50 μM I3C for 24 hours and then activated with 1 nM TNF for different times. Whole-cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by immunocomplex kinase assay. To examine the effect of I3C on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies.

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