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

After two washes with PBS, transfectod COS cells of one 60-ram plate were scraped into 1 ml PBS and centrifuged for 10 rain at 800 rpm (132 giv). The pellet was resuspended in 800/~1 MNT buffer (20 m_M MES, 30 mM Tris, 100 mM NaCl, 1.25 m_M EDTA, and 1 mM EGTA) at various pHs containing 1% Triton X-100, 100 mM iodoacetamide, 40 t~g/mi PMSF, and a 1:500 dilution of a protease inhibitor cocktail (5 mg/mi benzamidine and 1 mg/ml each of pepstatin A, leupeptin, antipain, and chymostatin in 40% dimedaylsnifoxide, 60 % ethanol) by passing it five times through a 25-gange needle connected to a 1-ml syringe. After a 1-h solubilization step on ice, the cells were centrifuged for 60 rain at 39,000 rpm (100,000 g~v) in a Ti 50 rotor (Beckman Instnmaents Inc., Paio Alto, CA). The resulting supernatants were carefully harvested and proteins were precipitated by the method of Wessel and Fluegge (1984) . The precipitates were solub'~,ed in Laemmli buffer (Laemmli, 1970) . The pellets in the Ti 50 tubes following centrifugation were selubilized directly in sample buffer and sonicated. Proteins were separated on 8% SDS-polyaerylamide minigels (Bio Rad Laboratories, Richmond, CA) using the Laemmli (1970) system, and transferred to nitrocellulose membranes according to the method of Towbin et al. (1979) . For the immunoreaction, the nitrocellulose sheet was blocked with 3 % nonfat dry milk powder in PBS, incubated with mAb G1/296 against p63 (diluted 1:5,000 in PBS-3% powdered milk) followed by a horseradish peroxidase-conjugated anti-mouse secondary antibody (Amersham Corp.). For development the ECL detection system (Amersham Corp.) was used according to the manufacturer's directions.