Skip to main content
. 2012 Feb 29;7(2):e32584. doi: 10.1371/journal.pone.0032584

Figure 5. Bortezomib-induced NF-κB activation.

Figure 5

Su-DHL8 cells were treated with 20 nM bortezomib with or without 50 µg/ml of CQ. (A and B) Effects of bortezomib or/and CQ on the levels of NF-κB proteins. Cytosolic and nuclear proteins were extracted from Su-DHL8 cells. 15 µg cytosolic or nuclear proteins were subjected to 4–20% NuPAGE gel. Anti-NF-κB RelA/p65, RelB, cRel, p50 and p52 rabbit antibodies were used at 1∶3000 dilution. Monoclonal Rb (the nuclear protein marker), and polyclonal LDH (the cytosolic protein marker) antibodies were used at 1∶2000 and 1∶500 dilution, respectively. Western blots presented are representative of 3 independent experiments performed. (B) Data analysis of NF-κB nuclear translocation. Significantly increased p65 or p50 protein levels after treatment with bortezomib (*P<0.05) was compared with their controls and analysed by t test (n = 3). Chloroquine-mediated inhibition on the nuclear translocation of RelA/p65, RelB, cRel and p50 was compared with their controls and treated with bortezomib alone. Significantly decreased nuclear protein levels were analyzed by t-test. (C) Effects of bortezomib or/and CQ on interaction between NF-κB and I-κBα. Dynabeads proteins A was coated with 2 µg of polyclonal anti-I-κBα antibody or normal rabbit IgG and then incubated with 300 µg protein. Interaction of I-κBα with NF-κB was determined by Western blotting with monoclonal anti-NF-κB antibody. IgG-Hv indicates IgG heavy chain (55 kD). Numbers under each pairs of bands are ratios of NF-κB/heavy chain. (D) Statistical data from three independent IP experiments analyzed by t test. (E) Effects of bortezomib or/and CQ on NF-κB RelA/65 DNA binding activity. Nuclear proteins were extracted using Nuclear Co-IP Kit. 10 µg nuclear proteins were used for the DNA binding assay. NF-κB DNA binding activity was determined using TransAM™ NF-κB p65 ELISA Kit. Data represent mean ± SD from 3 independent experiments. (F) Effects of bortezomib or/and CQ on NF-κB/cRel DNA binding activity. Su-DHL8 cells were treated with 20 nM bortezomib with or without 50 µM CQ for 6 hours. Protein-DNA complexes were extracted and bound with biotin-labeled NF-κB/cRel DNA binding site. Data represent two separate results from four independent experiments.