Mammalian two-hybrid analysis of interactions between N-CoR and RARα, PML-RARα, or PLZF-RARα and their ATRA sensitivities in vivo. Cotransfections of CV-1 cells with 125 ng of GAL(RE)5-tkluc reporter plasmid, 35 ng of GAL4(DBD)-N-CoR expression vector (or an empty vector), 100 ng of CMV-lacZ internal control, and 125 ng of an expression vector for a given VP16 fusion protein, as indicated, were performed in 24-well plates using calcium phosphate precipitation and approximately 105 cells per well. Where indicated, cells were treated 24 to 26 hours after transfection with 10−6 mol/L ATRA for approximately 20 hours before harvesting. Similar to the VP16-RARα control, cotransfection of an empty GAL4(DBD) vector (pGALO) either with VP16-PML-RARα or VP16-PLZF-RARα did not result in activation of the luciferase gene expression (not shown).
