FIG 5.

CITFA2 directly contacts the BES promoter and is required for CITFA to bind to RNA Pol I promoters in vivo. (A) Sucrose gradient fractions of purified CITFA, shown in Fig. 2A, were used in an EMSA with a radiolabeled BES promoter that was visualized by autoradiography. Fraction 12, which contains minimal LC8 and CITFA2 and an abundance of other CITFA subunits, barely binds to the probe (arrow), while fractions 13 and 14, which contain an abundance of all CITFA subunits, effectively bound the promoter probe. (B) UV cross-linking analysis using tandem affinity-purified CITFA with radiolabeled BES promoter. After DNA digest, proteins were separated by SDS-PAGE and visualized by autoradiography. On the right, tagged and untagged CITFA subunits are identified. As explained in the text, the band that did not shift is putatively CITFA1 (put. CITFA1). (C) Anti-CITFA3 ChIP assay in an smCITFA2 cell line which was either not induced or in which PTP-CITFA2 was silenced for 2 days. *, P < 0.05; **, P <0.01.