The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

Coverslips containing injected 3T3 cells were processed for immunofluores-cence microscopy as follows. 4-5 h after injection, cells were fixed with 3 % paraformaldehyde in PBS for 20 min on ice. After fixation, cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 2 min on ice. Cells were then washed with PBS and nonspecitic sites blocked with milk buffer (2% nonfat dry milk, 0.1% Tween 20, 40 mM K2HPO,,, 10 mM KI-I2PO4, 150 mM NaCI, and 0.01% NAN3) for 1-2 h at room temperature. All subsequent steps were performed at room temperature. First antibody incubations to detect epitope-tagged proteins were performed with an mAb, 12C.A5 0dndly provided by Dr. Richard Lerner, Scripps Clinic, La Jolla, CA), specific for the HA peptide tag (34) (at a 1:300 dilution of ascites fluid in 1% BSA in PBS). The detection of wildtype CD8 and CD8-eontaining chimeric proteins was performed with an mAb, OKT8 (kindly provided by Dr. Giovanni Migliaccio, Istituto di Ricerche di Biologia Molecolare, Pomezia, Italy), directed against the extracellular domain of CD8 (at a 1:100 dilution of culture supematant in milk buffer). First antibody incubations were performed for 40 min followed by extensive washing with milk buffer. Primary antibody reactivity was visualized with Texas red-conjugated goat anti-mouse IgG (Molecular Probes, Inc., Eugene OR) used at 10 tzg/ml in milk buffer. Cells were probed for 40 min and then washed extensively with milk buffer followed by two washes with 1% BSA in PBS.