β-globin mRNA levels are dictated by AUF-1 and YB-1 in erythroid cells, but not in nonerythroid cells that do not assemble the β-complex. (A) Coordinate depletion of AUF-1 and YB-1 decreases steady-state levels of β-globin mRNA in erythroid K562 cells. K562β cells were transfected with specific siRNAs targeting AUF-1, YB-1, both AUF-1/YB-1 (A/Y), or control lamin mRNAs. Cytoplasmic extracts were analyzed using factor-specific and control GAPDH Abs (Figure 5B). β-globin mRNA was assessed by quantitative RT-PCR relative to endogenous control β-actin mRNA. The level of β-globin mRNA in mock transfected cells was set at 100%. Bars depict the mean ± SE of 5 independent experiments. (B) β-globin mRNA levels are not significantly affected by coordinate depletion of AUF-1 and YB-1 in cells that express both factors but do not assemble a β-complex in vitro. Left: HeLaβ cells were transfected with siRNAs targeting both AUF-1 and YB-1 (A/Y) or control lamin mRNAs, and immunoblot conducted with Abs against AUF-1, YB-1, and GAPDH. Right: Levels of β-globin mRNA in siRNA-transfected HeLaβ cells were determined relative to internal control β-actin mRNA by quantitative RT-PCR. The level of β-globin mRNA in mock-transfected cells was set at 100%. Bars depict the mean ± SE of 3 independent experiments.