FIG. 6.

Sp1 and Sp3 bind to the GC motif in the IL-21R promoter in vitro. (A) Proteins purified by DNA affinity chromatography. Nuclear extracts from PI-stimulated (4 h) human PB T cells were subjected to DNA affinity chromatography. Eluates from WT or mutant (Mut5, as in Fig. 5A) DNA-conjugated beads were resolved by SDS-PAGE and silver stained. LC-MS and MALDI-TOF mass spectrometry were conducted on the 95-, 80-, and 40-kDa bands (arrows). The 95-kDa band is Sp1. (B) Sequence comparison of human and mouse IL-21R promoter-proximal region showed a conserved GGGCGGGGC motif. (C) EMSA using WT probe and recombinant Sp1, Sp3, or nuclear extracts (NE) from human PB T cells treated with PI or not treated. Antibodies to Sp1, Sp2, Sp3, Sp4, or unlabeled oligonucleotides containing the Sp1 consensus sequence at 25-, 50-, or 100-fold molar excess were preincubated with nuclear extract before adding WT probe, as indicated. (D) Nuclear extracts (15 μg) from anti-CD3-plus-anti-CD28-activated human PB T cells or mouse splenic T cells were blotted with anti-Sp1 or γ-tubulin (control). (E) Nuclear extracts (15 μg) were blotted with anti-Sp3 or γ-tubulin.