FIG. 8.
Inhibition of Brap2 by siRNA does not inhibit p21 expression but reduces cytoprotective activity of cytoplasmic p21. (A) Percentage of cytoplasmic p21-expressing cells in U937/CB6-p21 cells treated with zinc for 3 days in the presence of siRNA. U937/CB6-p21 cells were transfected with control or Brap2-targeting siRNA and then treated with 120 μM ZnSO4 for 3 days. The results from one of three experiments are shown. (B) Brap2 expression was reduced in the presence of siRNA. Cell lysates from U937/CB6-p21 cells treated with zinc for 3 days in the presence of siRNA were extracted. Cell lysates were immunoprecipitated (IP) with anti-Brap2 antibody and subjected to Western blotting (WB) with anti-Brap2. Whole-cell lysates were blotted with anti-p21 antibody, demonstrating significant expression induction of p21 in both transfectants. The percentage of cells that were CD14 positive is shown under the blot. The asterisk indicates a nonspecific band. (C) Inhibition of Brap2 expression by siRNA reduces resistance against hydrogen peroxide-induced apoptosis of differentiated U937/CB6-p21 cells. U937/CB6-p21 cells were transfected with either control or Brap2-targeting siRNA and were cultured with Zn for 3 days. Cells were treated with 300 μM hydrogen peroxide for 16 h. Dead cells were scored by fluorescence-activated cell sorting analysis. The results from one of three experiments are shown.