Figure 5

Functions of TFIIB and the acetylated TFIIB variant in cell cycle transitions.

(A) TFIIB immunofluorescence (red) after release from nocodazole block (hours). DNA is stained with DAPI (blue). (B) DAPI flow cytometry of control siRNA (wt) or TFIIB siRNA (−B) treated cells after release from nocodazole block (hours). DNA content is shown below (2N, 4N). (C) Confocal microscopy of nonacetylated and acetylated TFIIB in HeLa cells visualized with nonacetylated TFIIB specific (αB) or acetyl-TFIIB (αAcB) specific antibodies by immunofluorescence (red) with DAPI staining (blue) during the indicated above the plots cell cycle phase. (D) A western blot (left) shows that the TFIIB antibody (αB) in a mixture with the 12 amino acids acetyl Lysine TFIIB peptide (acp) recognises only the nonacetylated recombinant TFIIB (rhnB, lane 1) and not acetylated recombinant TFIIB (rhacB, lane 2). The anti acetyl-TFIIB andibody (acB) (right blot) recognises the acetylated recombinant protein (lane 4) and not the nonacetylated TFIIB protein (lane 3). Ponceau S (S) staining of each blot before immunostainning is shown beneath the respective westerns. The unprocessed images are shown in Supplemental Figure 6.