FIG. 6.

HIRA-induced S-phase arrest is overridden by the histone mRNA-stabilizing protein, SLBP. (a) U2OS cells were transiently transected with pcDNA3 (lanes 1 and 9) or pcDNA3 HA-HIRA WT (lanes 2 to 8) in the absence or presence of pcDNA3 HA-SLBP (0.01, 0.1, 0.25, 0.5, 1, or 3 μg [triangle]) together with pCMV-CD19. Total-cell lysates were prepared and Western blotted with antibodies to SLBP or HA-HIRA. (b) Same as panel a except that the cell cycle distribution of CD19⁺ cells was determined. (c) RNA was prepared from transfected CD19⁺ cells and Northern blotted to determine abundance of histone H2B. (d) U2OS cells were transiently transfected with pcDNA3 or pcDNA3 HA-HIRA WT in the absence or presence of pcDNA3 HA-SLBP (3, 8, or 18 μg [triangle]) together with pCMV-CD19. The cells were arrested in G₁ phase with mimosine and then released into S phase. The cell cycle distribution of the CD19⁺ cells was determined 12 h after release. (e) Cell lysates from panel d were Western blotted with anti-HA antibodies.