Gel-shift assay with HAF protein.

The probe used was the region −45 to −61 bp (EP17) of the epo promoter. (A) Gel-shift assay using a crude extract from HAF recombinant lysogen. The crude extract (CE) (5 μg) was incubated with radiolabel led EP17 alone (lane 1), or in the presence of a 500-fold molar excess of the specific competitor EP31 (lane 2) or nonspecific competitor EP22 (lane 3). (B) Gel-shift assay with HAF GST-fusion protein. Increasing amounts (5, 10, and 20 μg) of GST protein (lanes 2-4) or HAF GST-fusion protein (lanes 5-10) were incubated with 100 ng of poly dI-dC (lanes 5-7), or 500 ng of poly dI-dC (lanes 2-4 and 8-10). Lanes 11 and 12 represent binding reactions with 10 μg of GST-fusion protein in the presence of 100- and 500-fold molar excess of the specific competitor EP31. Lane 1 represents the binding reaction with no protein. Each of these reactions was carried out in the presence of 2 μg of BSA as carrier protein. The arrow indicates the shifted complex. F is free probe.