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. 2012 Apr 13;7(4):e34501. doi: 10.1371/journal.pone.0034501

Figure 3. Repression of Flt3 by N-CoR protein.

Figure 3

A, Relative activity of a luciferase reporter driven by the Flt3 promoter was determined in various leukemic cell lines. The cells were transfected with reporter and reference plasmids using electroporation. The values presented in each bar represent the average of three independent experiments. B, Effect of ectopic N-CoR on the activity of the Flt3 promoter in THP-1 cells electroporated with Flag-tagged N-CoR plasmid in a dose dependent manner was determined via luciferase assay (left panel). The values presented in each bar represent the average of three independent experiments. In parallel, levels of ectopic N-CoR protein in THP-1 cells used in the luciferase assay were determined in western blotting assay with anti-Flag antibody (right panel). C, Effect of ectopic N-CoR on the activity of the Flt3 promoter in 293T cells transfected with N-CoR or control siRNA was determined using luciferase assay. In pGL3-Flt3 (−901) reporter plasmid, luciferase reporter was placed under the control of the full length Flt3 promoter. The values presented in each bar represent the average of three independent experiments. D, The dose dependent fold repression by ectopic N-CoR in N-CoR ablated or non-ablated 293T cells was calculated by dividing the mean relative luciferase activity in N-CoR siRNA transfected cells with that of control siRNA transfected cells of Fig. 3C. E, N-CoR is associated with the Flt3 promoter. Relative amounts of Flt3 promoter sequence associated with N-CoR protein in HL-60 or NB4 cells were determined through ChIP assay. The antibody used in the ChIP assay is mentioned at the bottom. N-CoR association with CD36 promoter, a known N-CoR target gene, was determined as positive control.