Journal: International journal of oncology

Article Title: Quinone methide tripterine, celastrol, induces apoptosis in human myeloma cells via NF-κB pathway.

doi: 10.3892/ijo.2011.1161

Figure Lengend Snippet: Figure 5. Effects of celastrol on NF-κB activity in myeloma cells. (A) U266 cells were treated with 50 ng/ml TNF-α for 12 h, and then the cells were incubated with 0.5 µM of celastrol for 6 and 12 h. The DNA binding activity of NF-κB in U266 cells was quantified by ELISA with the use of a Trans-AM NF-κB p65 transcription factor assay kit. Celastrol significantly suppressed the stimulatory effect of TNF-α on NF-κB DNA binding activity (P<0.01). The control is referred to as no stimulated control, the wild-type as wild-type oligonucleotide, and the mutant as mutant oligonucleotide. (B) Effects of celastrol on the constitutive expression of NF-κB and IκB-α in myeloma cells. U266 cells were treated with celastrol (0.5 µM) for the indicated times. Cell lysates (15 mg protein per lane) were fractionated in 12.5% SDS-polyacrylamide gels and analyzed by Western blotting with an antibody against NF-κB p65 and IκB-α. Nuclear and cytoplasmic extracts were prepared in order to check these proteins by Western blotting.

Article Snippet: The level of NF-κB was assessed by ELISA using monoclonal antibodies and the procedure recommended by the manufacturer (TransAM NF-κB p65 transcription factor assay kits; Active Motif, Carlsbad, CA).

Techniques: Activity Assay, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Transcription Factor Assay, Control, Mutagenesis, Expressing, Western Blot