The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

All manipulations were done at room temperature. Cells were washed with PBS after each step of fixation and antibody incubation. Antibodies were diluted in 3% BSA/PBS. Transfected cells on coverslips were fixed for 3 rain in 3.7% formaldehyde and permeabilized for 5 rain with 0.5% Triton X-100. Cells were blocked for 15 rain in 3% BSA/PBS, followed by incubation for 60 rain with one of the following primary antibodies: affinitypurified rabbit anti-p58 IgG (I0 ~g/ml; described above); rabbit antiserum raised aplnst the small subunit of RuBPCase (l:100; a gift from Dr. Debkumar Pain), or fractionated escites fluid developed in mouse against &galactosidase (I:1000; Sigma Immunochemicais, St. Louis, MO), followed by a 15-rain incubation with the appropriate second antibody; goat anti-mouse or goat anti-rabbit IgG-FrIC (1:50; Cappel, Durham, NC). To measure the endogenous COS cell p58, human autoantibody (1:100; Courvalin et al., 1990) , followed by anti-human IgG-FITC (1:50; Bio-Rad Laboratories, Richmond, CA) was used. For the double immunotinorescence experiment, the two primary antibodies, affinity-purified anti-SSRc~ (4/~g/rnl; Migiiaccio et al., 1992) and a mAb to the HA tag (1:200; Berkeley Antibody Company, Richmond, CA) were incubated concomitantly as were the secondary antibodies, donkey anti-mouse IgG-FI'[C and donkey anti-rabbit IgG-Texas red (1:50; Jackson lmmunoResearch Labs, Inc., West Grove, PA). Coverslips were mounted in p-phanylenediamine solution (1 mg/ml) in 90% glycerol in PBS, pH 8.0 (Johnson and Nogueria Araujo, 1981) . Cells were viewed under an Axiophot (Carl Zeiss, Oberkochen, Germany) and photographed with T-MAX 400 Kodak film (Eastman Kodak Co., Rochester, NY).