A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

Indirect Immunofluorescence Microscopy COS-7 cells grown on coverslips were fixed, permeabilized, and stained 44 h posttansfection essentially as described (26, 27) . For detection of El Swift and Machamer Golgi Retention Signal and mutant El proteins, an affinity-purified rabbit anti-peptide antiserum recognizing the COOH-terminus of El was the primary antibody (1 :40, N5 gg/ml), followed by Texas red-conjugated, affinity-purified goat anti-rabbit IgG (1 :500 ; Jackson Immuno Research Laboratories Inc., Avondale, PA) . For detection of G protein and mutant G proteins by double labeling, nonpermeabilized fixed cells were first stained with a rabbit antiVSV serum (1 :200) followed by Texas red-conjugated, affinity-purified goat anti-rabbit IgG. After permeabilization with 0.5 % Triton X-100, internal G protein was detected by staining with a monoclonal anti-G antibody (11, 4 pg/ml; reference 23), followed by fluorescein-conjugated affinity-purified goat antimouse IgG (1 :200, Jackson Immuno Research Laboratories Inc.) . Cells expressing the chimeric am proteins were stained with an affinity-purified rabbit anti-peptide antibody which recognizes the G cytoplasmic tail (1 :20; reference 29) followed by the Texas red-conjugated second antibody described above . Cells were visualized with a Nikon Microphot microscope (Nikon Inc ., Garden City, NJ) equipped with epifluorescence illumination and a Nikon 60x oil immersion plan apochromat objective . Photographs were taken with Tri-X Pan film (Eastman Kodak Co ., Rochester, NY) and processed with Diafine developer (Accufine, Inc., Chicago, IL) .