Nuclear protein binding analysis of the RH enhancer fragment by DNase I protection assays.

The noncoding strand of the RH fragment was labeled with³²P at the 3′ end by an end-fill reaction. DNase I footprint analysis was carried out using 15 μg nuclear extract from HA-1 cells incubated with MPs. The DNase I digestion products were coelectrophoresed with the G+A chemical sequencing ladder of the RH probe on a denaturing 6% polyacrylamide gel and autoradiographed for 48 hours. Lane G+A is G+A chemical sequencing ladder; lane RH, RH probe alone digested with DNase I; lane BSA, RH probe incubated with BSA prior to DNase I digestion; lane ZC, nuclear extract from ZnCl₂-treated cells incubated with RH probe prior to DNase I digestion; lane SP, nuclear extract from SnPP-treated cells incubated with RH probe prior to DNase I digestion; lane ZP, nuclear extract from ZnPP-treated cells incubated with RH probe prior to DNase I digestion. The region protected from DNase I digestion is indicated by the arrows and the 9-bp sequence, which has 78% homology with Egr-1 is indicated with the parentheses.