FIG. 1.

TLF and TBP have different tissue and subnuclear localizations. (A) Western blot showing the presence of TLF in heart (He), kidneys (Ki), liver (Li), lungs (Lu), pancreas (Pa), and testes (Te) from an adult mouse. A longer exposure showed that TLF was also present in the brain (Br). (B) Western blots showing distinct amounts of TLF and TBP relative to TFIIB in various cell lines. Equal amounts of proteins from HEK-293, HeLa, and SK-N-SH cells were loaded. Parallel blots were stained for TFIIB, TLF, or TBP. HEK-293 cells have a lower TLF/TBP ratio than either SK-N-SH or HeLa cells. The amount of protein in each lane was normalized by a BCA protein assay and verified by Ponceau S staining. (C) eGFP does not localize to the nucleus. The upper cell, including the nucleus, is completely filled with eGFP (stained with Hoechst dye). The lower cell was untransfected, allowing visualization of the nucleus. (D) TLF-GFP localizes to the nucleus. HEK-293 cells were cotransfected with RFP and TLF-GFP. RFP fills the cell, while TLF-GFP is restricted to the nucleus. (E through H) TBP-GFP and TLF-GFP have different subnuclear localizations. Arrows mark the positions of the nucleoli. (E and F) Transmission differential interference contrast (DIC) and fluorescence images of COS-7 cells transfected with a TBP-GFP fusion construct. (G and H) Transmission DIC and fluorescence images of COS-7 cells transfected with a TLF-GFP fusion construct. (I and J) High-resolution images showing that TBP-GFP is excluded from, while TLF-GFP is concentrated in, the nucleoli. (Inset) Similar nucleolar exclusion in HEK-293 cells. Scale bars, 10 μm.