Figure 2

p300ΔC-820 localizes to the nucleus, but only weakly enhances REL transactivation. (a) Anti-p300 Western blotting was performed on extracts from control A293T cells, p300ΔC-820-transfected A293T cells, and SUDHL2 cells. Twenty-five μg of control A293T and SUDHL2 total protein and 30 ng of p300ΔC-820-transfected A293T total protein were loaded. p300ΔC-820-transfected A293T and SUDHL2 extracts were loaded together in the last lane. (b) DF-1 chicken fibroblasts were transfected with 3 μg of pCMVβ-p300, pCMVβ- p300ΔC-1087, or pCMVβ- p300ΔC-820 as indicated. Indirect immunofluorescence was performed using a primary anti-p300 antiserum and a FITC-conjugated anti-rabbit secondary antibody (top row). Nuclei were visualized by DAPI staining (bottom row). (c) A293 cells were co-transfected with 0.5 μg of pcDNA-REL along with 0.5 μg of pCMVβ-p300, pCMVβ-p300ΔC-1087, or pCMVβ-p300ΔC-820 as indicated. Western blotting shows relative p300 and REL expression in transfected cells (bottom panel). Luciferase and β-galactosidase activities were determined, and values were normalized to control transfections (1.0, top panel). Error bars represent standard error of the mean. Asterisks represent p-value < 0.05 for the difference between the indicated value and the control value (1.0) in a one-tailed t-test. (d) A293 cells were co-transfected with 0.5 μg of pcDNA-REL and the indicated amounts of pCMVβ-p300, pCMVβ-p300ΔC-820, or pCMVβ-p300ΔC-1087. Luciferase and β-galactosidase activities were determined, and values were normalized to REL-alone control transfections as indicated (1.0, white bar). Total DNA within each experiment was kept constant by the addition of empty pcDNA3.1 vector. For all reporter assays, values are the averages of at least three experiments, each performed with duplicate or triplicate samples. Error bars represent standard error of the mean.