American Society of Hematology

Figure 4

$Figure 4. Anacardic acid inhibits TNF-dependent IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. (A) Anacardic acid inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for the indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of anacardic acid on TNF-induced IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and treated with 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts (CE) and nuclear extracts (NE) were prepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using the indicated antibody. An anti–β-actin antibody was the loading control. (C) Effect of anacardic acid on the phosphorylation of IκBα by TNF. Cells were preincubated with 25 μmol/L anacardic acid for 4 hours, incubated with 50 μg/mL ALLN for 30 minutes, and then treated with 0.1 nmol/L TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using a phospho-specific anti-IκBα antibody. The same membrane was reblotted with anti-IκBα antibody. (D) Anacardic acid inhibits TNF-induced IκBα kinase activity. Whole-cell extracts were immunoprecipitated with antibody against IKKα and analyzed by an immune complex kinase assay. To examine the effect of anacardic acid on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKKα and anti-IKKβ antibodies. (E) Direct effect of anacardic acid on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNF and immunoprecipitated with anti-IKKα antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentration of anacardic acid. (F) Effect of anacardic acid on TNF-induced acetylation of p65. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF. Whole-cell extracts were prepared, immunoprecipitated with an anti-p65 antibody, and subjected to Western blot analysis using an anti–acetyl-lysine antibody. The same blots were reprobed with anti-p65 antibody. (G) Effect of anacardic acid on TNF-induced protein acetylation. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF for 20 minutes. Whole cell extracts were prepared and subjected to Western blot analysis using an anti–acetyl-lysine antibody. (H) Immunocytochemical analysis of p65 localization. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 15 minutes. Cells were subjected to immunocytochemical analysis.$

Anacardic acid inhibits TNF-dependent IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. (A) Anacardic acid inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for the indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of anacardic acid on TNF-induced IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and treated with 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts (CE) and nuclear extracts (NE) were prepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using the indicated antibody. An anti–β-actin antibody was the loading control. (C) Effect of anacardic acid on the phosphorylation of IκBα by TNF. Cells were preincubated with 25 μmol/L anacardic acid for 4 hours, incubated with 50 μg/mL ALLN for 30 minutes, and then treated with 0.1 nmol/L TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using a phospho-specific anti-IκBα antibody. The same membrane was reblotted with anti-IκBα antibody. (D) Anacardic acid inhibits TNF-induced IκBα kinase activity. Whole-cell extracts were immunoprecipitated with antibody against IKKα and analyzed by an immune complex kinase assay. To examine the effect of anacardic acid on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKKα and anti-IKKβ antibodies. (E) Direct effect of anacardic acid on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNF and immunoprecipitated with anti-IKKα antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentration of anacardic acid. (F) Effect of anacardic acid on TNF-induced acetylation of p65. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF. Whole-cell extracts were prepared, immunoprecipitated with an anti-p65 antibody, and subjected to Western blot analysis using an anti–acetyl-lysine antibody. The same blots were reprobed with anti-p65 antibody. (G) Effect of anacardic acid on TNF-induced protein acetylation. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF for 20 minutes. Whole cell extracts were prepared and subjected to Western blot analysis using an anti–acetyl-lysine antibody. (H) Immunocytochemical analysis of p65 localization. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 15 minutes. Cells were subjected to immunocytochemical analysis.

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