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

For double-immunofluorescence experiments with the NPC-specific mAb 414 (7), the expressed recombinant protein was first detected with primary and secondary antibodies as described above and then processed as follows. Ceils were washed with PBS, refixed with 1% paraforrnaldehyde in PBS for 1 min, and washed extensively with PBS. Samples were then washed once with milk buffer and incubated for 15 min with a 1:50 dilution of mouse serum in milk buffer to block nonspecific sites. After this incubation, cells were probed for 30 rain with FITC-labeled mAb 414 (kindly provided by Dr. Thomas Meier, Rockefeller University, New York, NY) in milk buffer containing a 1:100 dilution of mouse serum. Unbound antibody was removed by extensive washing with milk buffer followed by two washes with 1% BSA in PBS. For all immunofluorescence experiments, cells were mounted in 90% glycerol containing 1 mg/ml p-phenylenediamine and viewed with a Zeiss Axiophot microscope. Photographs were taken through a 63x or 100x objective onto Kodak (T-Max 400, Eastman Kodak Co., Rochester, NY) film push processed to 1600 ASA.