FIG. 3.

HMG-1 and HMG-2 purification and effects on PR-DNA binding in vitro. (A) HMG-1 and HMG-2 from calf thymus were purified and separated on a PBE94 chromatofocusing column as described in Materials and Methods. Coomassie blue-stained SDS-polyacrylamide gels (12%) and Western blot analysis of purified HMG-1/-2 fractions were prepared as follows: lane 1 (pool 1), HMG-2; lane 2 (pool 2), HMG-1 and HMG-2; lane 3 (pool 3), HMG-1; lane 4, purified recombinant polyhistidine-tagged HMG-1. Western blots were done with an IgM MAb (854/E10) that recognizes both HMG-1 and HMG-2. (B and C) The influence of purified calf thymus HMG-1 (pool 3) and HMG-2 (pool 1) on the binding of purified PR-A to a 32-bp PRE oligonucleotide probe as assessed by EMSA. A constant amount (30 nM) of purified PR-A was incubated with increasing amounts of purified HMG-1 (B) or HMG-2 (C). The amounts of HMG-1 and HMG-2 used were 30, 50, 70, 100, 200, 300, 400, 500, 700, and 1,000 μg/assay in lanes 3 to 12, respectively.