Figure 2: Sam68 is a novel p65-interacting protein in the NF-κB complex binding to the CD25 κB site.

(a) The cell surface CD25 expression in resting Jurkat A3 cells and HUT102 cells was stained with isotype antibody or PE-conjugated CD25 antibody, and analysed by FACS. (b) Nuclear extracts from HUT102 cells were immunoblotted (IB) directly or after immunoprecipitation (IP) with isotype (iso) or p65 antibody for Sam68. (c) The purification scheme for proteins specifically recognizing CD25 κB DNA by gel filtration and DNA affinity purification in the HUT102 cells. (d) Nuclear extracts prepared from HUT102 cells were separated on a Superose 6 gel filtration column. The fractions 14–36 were interrogated for CD25 κB DNA-binding activity in EMSAs. The unfractionated nuclear extract (NE) was also analysed with ³²P-labelled CD25 κB probe or with 100-fold unlabelled CD25 κB oligonucleotide competitors (CC). The CD25 κB complex was indicated by a triangle and the elution positions of protein standards are indicated. (e) The Superose 6 fractions 14–36 were immunoblotted (IB) with p65, p50, and Sam68 antibodies and the elution positions of protein standards are indicated. NE indicates the unfractionated nuclear extract. (f) Schematic diagram of vector double-stranded DNA or that contains five copies of CD25 κB sites. Immunoblot (IB) of the elution after DNA affinity purification for indicated proteins from pooled fractions from 24 to 30 harboring high CD25 κB DNA-binding activity.