Figure 2.

Expression of β-globin by the CAGGS promoter in non-erythroid cells. (A) Western blot analysis of cell extracts from U87, Daoy, HCT116 and MCF7 cells transiently transfected with pCAGGS-β3′Δ plasmid. The cells transfected with the β-globin construct were collected 48 h after the addition of hemin (20 μM) and the western blot analysis was performed as described in “Material and Methods.” β-actin was used to control for lane loading. (B) The transfection efficiency in U87, Daoy, HCT117, and MCF7 cells was determined by parallel transfection with the pCAGGS-DsRed2 plasmid. (C) β-globin protein undergoes proteasomal degradation in non-erythroid cells. Western blot analysis of HuH-7 (left), or Daoy and MCF7 cells (right) transiently transfected with pCAGGS-β3′Δ plasmid. Twenty-four h post-transfection, the cells were first induced with hemin (20 μM) and then MG132 was added at the indicated concentrations (top) after an additional 24 h. The cells were harvested and processed for western blot analysis 72 h after transfection. Control, no transfection control for HuH-7. (D) β-globin transcript levels in erythroid and non-erythroid cell lines. Reverse-transcribed (RT) PCR analysis of β-globin mRNA levels from pCAGGS-β3′Δ in the presence or absence of hemin. The cell line is indicated above and β-actin transcript levels were used as an internal standard to verify RNA quality. The 500 bp DNA maker is shown at left with the transcript and its respective size indicated at right.