Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers

COS cells (African green monkey kidney cells, CRL 1650; American Type Culture Collection, Rockville, MD) were cultured in DME supplemented with 10% FCS, 50 units/ml penicillin, 50 t~g/ml streptomycin, and fungizone at 37°C in a humidified 5% CO: atmosphere. For transient tr~ons COS cells were plated at 17,500-35,000 cells per well in an eight-well multichamber slide in which the wells had been coated for at least 20 rain with 25 tLg/ml fibronectin in PBS, or at 106 cells per 60-mm plate. Suboonfluent cells were washed once with TBS and each 60-ram plate was exposed to 3/~g of DNA in 1.5 ml RPMI-1640 medium containing 10% Nnsera, 50 t~g/ml gentumycin, 100 t~M ehloroquiue, and 150/~g/ml DEAE-dextran (RNC plus DEAE-dextran) for 3.5 h. For eightwell multichamber slides 0.2/~g DNA in 100/~1 RNC plus DEAE-dextran was used in each well. The DNA solution was then replaced with PBS containing 10% DMSO, cells were incubated for 2 rain at room temperature, and washed with PBS before fresh medium was added. Cells were maintained at 37"C and analyzed 43-48 h after transfection.