The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

Transfected cells were grown to 60% confluency on 35-rnm-diameter petri dishes. Cells were washed three times with PBS, fixed with 3 % formaldehyde/PBS for 30 min and washed three times with PBS/0.1% Triton X-100 (solution A). Cells were then incubated with PBS/0.1% Triton X-100/2% BSA (solution B) for 60 min and then for 90 min with the appropriate antibody at a 1:100 dilution in solution B. Cells were then washed five times with solution A, incubated for 60 min with a 1:100 dilution of fluoresceinlabeled donkey anti-rabbit IgG (Amersham, Arlington Heights, IL) in solution B plus 2 tLg/ml 4,6-diamidino-2-phenylindole (DAPI), and again washed five times with solution A. Coverslips were mounted with a 2% wt/vol solution of 1,4-diazabicyclo-(2.2.2)-octane in 85% glycerol/15% 8 mM Tris-HC1 (pH 8.6). Immunofluorescence microscopy was performed on a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY). T-MAX 3200 or T-MAX 400 film (Eastman Kodak Co., Rochester, NY) were used for photography.