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 (10-12 60-mm plates per preparation) transfected with either p63wt or A2-101AA were washed twice with PBS, scraped into 1 ml PBS per plate and centrifuged for 10 min at 800 rpm (132 ga~). Each pellet was resuspended in 500/~1 of buffer 2 (100 mM Na2HPO4, pH 8.0, 1% Triton X-100), containing 100 mM iodoacetamide, 40/~g/ml PMSF, and a 1:500 dilution of the above described protein inhibitor cocktail by passing it five times through a 25-gang¢ needle connected to a 1-ml syringe. After a 1-h solub'dization step on ice, the cells were centrifuged for 60 rain at 39,000 rpm (100,000 gay) in a Ti 50 rotor. P63 proteins were affinity purified from the resulting supernatants on a column of mAb G1/296 coupled to cyanogen bromide-activated Sepharose 4B. mAb G1/296 had been purified from culture supernatant by ammonium sulfate precipitation (to 50 % saturation) followed by protein A-Sepharose chromatography prior to coupling according to the suppliers instructions. P63 proteins were eluted with 0.1 M glycine, pH 3.0, 0.05 % Triton X-100, and immediately adjusted to pH 8.0 with 1 M "Iris base. After analysis by SDS-PAGE, fractions that revealed a single band of appropriate size (Schweizer, A., J. Rohrer, and S. Kornfeld, unpublished data) were pooled and dialyzed against MNT buffer, pH 8.0, containing 0.05% Triton X-100. Protein was determined with the micro BCA protein assay (Pierce, Rockford, IL).