American Society of Hematology

Figure 2

$Figure 2. Gossypin inhibits TNF-induced NF-κB activation, IκBα degradation, IκBα phosphorylation, IKK activation, p65 nuclear translocation, and p65 phosphorylation. (A) Gossypin inhibited time-dependent TNF-induced activation of NF-κB. KBM-5 (2 × 106) cells were preincubated with 50 μM gossypin for 2 hours. They were then treated with 0.1 nmol/L TNF for the indicated times and analyzed for NF-κB activation by EMSA. (B) Effects of gossypin on TNF-induced degradation of IκBα. KBM-5 cells (2 × 106) were preincubated with 50 μM gossypin for 2 hours for the indicated times. Cytoplasmic extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using anti- IκBα. (C) Gossypin suppressed TNF-induced phosphorylation of IκBα. KBM-5 cells (2 × 106) were preincubated with 50 μM gossypin for 2 hours. N-acetyl-Leu-Leu-norleucinal was added, and the cells were treated with 0.1 nmol/L TNF for 15 minutes. Cytoplasmic extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using antiphospho-specific IκBα and anti-IκBα. (D) Gossypin suppressed the TNF-induced activation of IKK. KBM-5 cells were pretreated with 50 μM gossypin for 2 hours and then treated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IKK-α and analyzed by immune complex kinase assay, as described in “Materials and methods.” To determine the effect of gossypin on the level of IKK proteins, whole-cell extracts were fractionated by SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies. (E) Immunocytochemical analysis of p65 localization. KBM-5 cells (2 × 106) were preincubated with 50 μM gossypin for 2 hours, TNF for 15 minutes, and subjected to immunocytochemical analysis, as described in Materials and methods. (F,G) Effects of gossypin on TNF-induced phosphorylation of p65. KBM-5 cells (2 × 106) were preincubated with 50 μM gossypin for 2 hours and then treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using phosphospecific p65.$

Gossypin inhibits TNF-induced NF-κB activation, IκBα degradation, IκBα phosphorylation, IKK activation, p65 nuclear translocation, and p65 phosphorylation. (A) Gossypin inhibited time-dependent TNF-induced activation of NF-κB. KBM-5 (2 × 10⁶) cells were preincubated with 50 μM gossypin for 2 hours. They were then treated with 0.1 nmol/L TNF for the indicated times and analyzed for NF-κB activation by EMSA. (B) Effects of gossypin on TNF-induced degradation of IκBα. KBM-5 cells (2 × 10⁶) were preincubated with 50 μM gossypin for 2 hours for the indicated times. Cytoplasmic extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using anti- IκBα. (C) Gossypin suppressed TNF-induced phosphorylation of IκBα. KBM-5 cells (2 × 10⁶) were preincubated with 50 μM gossypin for 2 hours. N-acetyl-Leu-Leu-norleucinal was added, and the cells were treated with 0.1 nmol/L TNF for 15 minutes. Cytoplasmic extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using antiphospho-specific IκBα and anti-IκBα. (D) Gossypin suppressed the TNF-induced activation of IKK. KBM-5 cells were pretreated with 50 μM gossypin for 2 hours and then treated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IKK-α and analyzed by immune complex kinase assay, as described in “Materials and methods.” To determine the effect of gossypin on the level of IKK proteins, whole-cell extracts were fractionated by SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies. (E) Immunocytochemical analysis of p65 localization. KBM-5 cells (2 × 10⁶) were preincubated with 50 μM gossypin for 2 hours, TNF for 15 minutes, and subjected to immunocytochemical analysis, as described in Materials and methods. (F,G) Effects of gossypin on TNF-induced phosphorylation of p65. KBM-5 cells (2 × 10⁶) were preincubated with 50 μM gossypin for 2 hours and then treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared, fractionated by 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. A Western blot analysis was performed using phosphospecific p65.

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