Figure 2

Modulation of NF-κB DNA binding activity by CCN2. HSC were harvested at the desired time points after treatment with or without 100 ng/ml CCN2. 6 μg nuclear protein extract were used in 20 μl reactions, containing 0.2 ng ³²P-labeled double strand NF-κB oligonucleotides. Reactions were fractioned through a nondenaturing 4% polyacrylamide gel. (A) Complex 1 (p65/p50) and complex 2 were enhanced after stimulation with CCN2. "Ctrl" represents a reaction lacking nuclear extract. (B) Bay11-7082 inhibited complex formation when added prior to CCN2 treatment ("Bay 11 + CCN2") but not when added subsequent to a 1 hour pretreatment with CCN2 ("CCN2 + Bay11"). (C) A supershift assay was performed by incubating pre-assembled gel shift assay complexes containing 8 μg nuclear extract with either 2 μg normal rabbit IgG or 2 μg anti-NF-κB antibody prior to separation through 8% polyacrylamide gel, showing that CCN2 stimulates the formation of an anti-p65/p65/anti-p50/p50/NF-κB oligonucleotide (S1) and an anti-p65/p65/NF-κB oligonucleotide (S2) supershift complexes. (D) A gel shift assay was performed following pre-incubating the nuclear extracts with either ³²P-labeled p50/p65 site mutant oligonucleotides or CBF-1 site mutant oligonucleotides.