Figure 8. A minimum cytoplasmic tail is required for stable membrane association.

a) 3T3 cells transiently expressing GFP-B7-38, GFP-B7-5 or GFP-B7-1 were stained with DAPI (blue) and then observed under a fluorescence microscope. The lower panels show a higher magnification view. b) 10⁷ 3T3 cells or stable 3T3 transfectants expressing AFP-B7-38, AFP-B7-5 or AFP-B7-1 were lysed in 200 µl carbonate buffer and centrifuged at 100,000xg. The insoluble fraction from 2x10⁶ cells (upper panel) or carbonate soluble fraction from 5x10⁶ cells (lower panel) were separated by SDS PAGE and then immunoblotted with anti-HA antibody to visualize AFP chimeric proteins. c) 3T3 cells stably expressing AFP-B7-38, AFP-B7-5 or AFP-B7-1 were solubilized and immunoprecipitated with anti-AFP antibody. One tenth of the mixture, corresponding to 10⁶ cells, was separated by SDS PAGE and immunoblotted with anti-HA antibody to visualize AFP chimeric proteins (upper panel). The remaining lysate was separated by SDS PAGE and then immunoblotted with rabbit anti-BiP (GRP78) antibody (middle panel). The lower panel shows an immunoblot for total BiP in whole cell lysates from 5x10⁵ cells.