American Society of Hematology

Figure 5

$Figure 5. Effect of NF-κB inhibition on Aiolos expression. (A) Analysis of NF-κB DNA-binding activity in B cells from CLL. Electrophoretic mobility shift assays were performed with total extracts from PBMC of 7 CLL patients (P13, P20, and P32-P36) and 3 HD using a 32P-labeled human immunodeficiency virus-long terminal repeat tandem κB oligonucleotide as a probe. (B) For supershift analysis, total extracts from 2 CLL patients (P35 and P32) were incubated with the indicated antibodies before incubation with the labeled probe. (C) Analysis of NF-κB DNA-binding activity in Daudi cells. Electrophoretic mobility shift assays were performed with nuclear extracts from Daudi cells in 3 independent experiments. Daudi cells were treated for 8 hours with the NF-κB inhibitor BAY 11-7082 at 5μM (referred as BAY, left) or for 2 hours with the wild-type NEMO binding domain peptide at 20μM (referred as NBD, right). DMSO or mNBD was used as a control. (D-E) Expression of Aiolos and antiapoptotic molecules in NF-κB inhibitor-treated Daudi and CLL cells. The expression of different genes was analyzed by qRT-PCR and normalized by use of the 2−ΔCt calculation method (Ct indicates cycle threshold) and β-actin as reference gene. Daudi and CLL cells were treated with BAY 11-7082 (5μM, 8 hours; panels D-E left) or NBD peptide (20μM, 2 hours; panels D-E right). CLL cells from 2 (P1 and P37) and 4 (P1 and P38-40) patients were treated with BAY and NBD, respectively. DMSO or mNBD was used as controls. Results are expressed as fold down- or up-regulation of inhibitor-treated cells compared with control cells. Data are mean ± SEM of at least 3 independent experiments. (F) Aiolos expression at the protein level was analyzed by Western blotting in nuclear (N) or cytosolic (C) extracts isolated from Daudi cells treated or not with BAY (5μM, 8 hours). Fibrillarin and β-actin expression were used as internal control of the nuclear and cytosolic fraction purity, respectively. Densitometric analysis of nuclear proteins and molecular weight of the proteins are shown. Similar results were obtained in 2 independent experiments.$

Effect of NF-κB inhibition on Aiolos expression. (A) Analysis of NF-κB DNA-binding activity in B cells from CLL. Electrophoretic mobility shift assays were performed with total extracts from PBMC of 7 CLL patients (P13, P20, and P32-P36) and 3 HD using a ³²P-labeled human immunodeficiency virus-long terminal repeat tandem κB oligonucleotide as a probe. (B) For supershift analysis, total extracts from 2 CLL patients (P35 and P32) were incubated with the indicated antibodies before incubation with the labeled probe. (C) Analysis of NF-κB DNA-binding activity in Daudi cells. Electrophoretic mobility shift assays were performed with nuclear extracts from Daudi cells in 3 independent experiments. Daudi cells were treated for 8 hours with the NF-κB inhibitor BAY 11-7082 at 5μM (referred as BAY, left) or for 2 hours with the wild-type NEMO binding domain peptide at 20μM (referred as NBD, right). DMSO or mNBD was used as a control. (D-E) Expression of Aiolos and antiapoptotic molecules in NF-κB inhibitor-treated Daudi and CLL cells. The expression of different genes was analyzed by qRT-PCR and normalized by use of the 2^−ΔCt calculation method (Ct indicates cycle threshold) and β-actin as reference gene. Daudi and CLL cells were treated with BAY 11-7082 (5μM, 8 hours; panels D-E left) or NBD peptide (20μM, 2 hours; panels D-E right). CLL cells from 2 (P1 and P37) and 4 (P1 and P38-40) patients were treated with BAY and NBD, respectively. DMSO or mNBD was used as controls. Results are expressed as fold down- or up-regulation of inhibitor-treated cells compared with control cells. Data are mean ± SEM of at least 3 independent experiments. (F) Aiolos expression at the protein level was analyzed by Western blotting in nuclear (N) or cytosolic (C) extracts isolated from Daudi cells treated or not with BAY (5μM, 8 hours). Fibrillarin and β-actin expression were used as internal control of the nuclear and cytosolic fraction purity, respectively. Densitometric analysis of nuclear proteins and molecular weight of the proteins are shown. Similar results were obtained in 2 independent experiments.

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