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Fig. 4. N-CoR is translocated to the cytoplasm in the presence of p65{Delta}TA. (A) Immunofluorescence staining of 293T cells transfected with N-CoR in the presence (lower panels) or absence (upper panels) of p65{Delta}TA. N-CoR-flag was detected by incubating with {alpha}-flag antibody and a FITC-conjugated secondary antibody (left panels), {alpha}-p65 staining is represented in the middle panels corresponding to Cy3 signal and nuclei were visualized by DAPI staining (right panels). Ectopic expression of p65{Delta}TA does not affect N-CoR protein levels. (B) Immunoblotting analysis of whole cell extracts isolated from 239T cells transfected with N1-IC and N-CoR-flag (lane 1) and N1-IC, N-CoR-flag and p65{Delta}TA (lane 2). Cell lysates were separated on a 6% acrylamide gel, and immunoblots were performed with the following antibodies: {alpha}-flag (N-CoR), {alpha}-N1(N1-IC) and {alpha}-p65. Protein loading for each lane was normalized according to ß-galactosidase expression and confirmed with {alpha}-N1 immunoblot. The western blot for p65 shows endogenous p65 (upper band) and transfected p65{Delta}TA (lower band).