Determination of the molecular mass of the Jurkat nuclear factor that binds the +18/+39 region of the CD43promoter.

(A) EMSA analysis performed as described in Figure 3 except binding reactions contained the radiolabeled oligonucleotide CD43 PyRo SSUB, which is both bromouracil- and biotin-modified, and either no protein extract (Probe) or a nuclear extract prepared from Jurkat cells. Binding reactions containing Jurkat nuclear extract were performed in the absence (−) or presence (+) of a 100-fold molar excess of unlabeled CD43 PyRo SS56 or the presence of the unrelated oligonucleotide NS-SS (NS). Marked with arrows are the positions of migration through a native polyacrylamide gel of CD43 PyRo SSUB unbound by protein (Free Probe) and bound by hnRNP-K. This analysis established that the protein-binding characteristics of CD43 PyRo SSUB are indistinguishable from its equivalent CD43 PyRo SS, which is modified neither with bromouracil nor biotin56. (B) The oligonucleotide CD43 PyRo SSUB was radiolabeled, incubated with (−) or without (Probe) a nuclear extract prepared from Jurkat cells and then exposed to UV light. CD43 PyRo SSUB was captured by streptavidin-coated magnetic particles, washed, and subjected to electrophoresis through a 12% SDS-polyacrylamide gel and autoradiography.