Figure S1. Characterization of protein/DNA complexes by specific antibodies to p50 or p65
NF-B subunits.
U937 cells were either treated with vehicle or treated with 128 µM AZT and after 6 h the NF-B
complex activation was assayed by non-radioactive EMSA. To characterize protein/DNA
complexes polyclonal antibodies to p50 or to p65 were added to nuclear extracts and samples were
incubated for 30 min on ice before the binding reaction. Moreover, to further confirm that the
observed shifted bands were specific for NF-B, additionally to the labeled NF-B probe a 200X
excess of unlabeled DNA probe was added during the binding reaction. Addition of p50 specific
antibody caused a supershift and a decrease in the intensity of the primary shifted signal that was
upregulated by AZT treatment, while addition of p65 specific antibody caused only a clear decrease
in the intensity of the primary shifted signal. The unlabeled competitor completely abolished the
visualization of the bands. These results confirm the NF-B specificity of the activated signal and
indicate the involvement of both p65 and p50 subunits in the activated complex. A representative
experiment out of the four performed with similar results is shown.