Figure 4

Preparation of endogenous and recombinant Bright. (A) Bright expression in human B cell lines. Crude nuclear extracts were prepared from the indicated B cell lines (lanes 2–9), from murine embryonic fibroblasts (MEFs) (-, lane 9) or from MEFs transduced with retroviral HA-Bright (+, lane 1). Approximately 15 μg/lane were fractionated on SDS-PAGE, and proteins were identified by western blotting with either an anti-ubc9 mAb (loading control, lower panel) or an affinity purified polyclonal rabbit anti-Bright antibody [21] (upper panel). (B) Purification from bacteria and analyses of Lac-inducible, His-tagged Bright (177–601). Left panel: SDS-PAGE/silver stain assay for purification after each step. Affinity chromatography on Ni beads (Affinity, lane 2) was followed by DEAE biogel agarose chromatography (DEAE, lane3). DNA affinity chromatography employing a Sepharose-conjugated, high-affinity Bright binding P2 site trimer [66] produced high yield and purification (not shown). But we were incapable of preventing the protein (grown in DH5α) from degradation to ~20 kD (Affinity 2X, lane 4) unless the plasmid was transformed into K1309 [44], a strain over-producing chaperones groE and groF (Affinity 2X+Chap, lane 5). Right panel: Specificity of P2 site-containing MAR binding of Bright (177–601) as judged by EMSA/competition. Lane 1, nuclear (N) extract prepared from BJAB B cells; lane 3, HA-Bright prepared from retrovirally transduced MEF nuclear extract; lanes 5 and 6, Bright (177–601) purified from E coli (protein inputs correspond to lanes 5 and 4, respectively, of left panel); lanes 2, 4, and 7, competition (of the corresponding protein sources (indicated by identically colored boxes at top of lanes) with ~150-fold molar excess of a P2 site-containing duplexed oligonucleotide [66]. The endogenous NF-μNR negative regulator [12,66] which binds to the same P sites as Bright (Fig. 1B) is the slower mobility complex indicated in lane 3.