CONFOCAL FLUORESCENCE MICROSCOPY FOR ANTIBODIES AGAINST A HIGHLY CONSERVED SEQUENCE IN SH2 DOMAINS

We have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SR2 domains. Mouse IgG1s (12E and 32D) prepared aga inst a peptide-conjugated keyhole lympet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analy sis showed that one IgG1 (12E) recognized mainly 62 kDa proteins (poss ibly src-family tyrosine kinases) from triton X-100 extracts of RBL-2H 3 cells and that another (32D) recognized mainly 32 and 110 kDa protei ns. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribu tion was observed in the cells stained with 12E and 32D IgGs. 12E IgGs strongly stained the plasma membranes while 32D IgGs stained small gr anules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggest that tyrosine kinases cluster along the plasma membranes and/or that conformational changes occur in the domai ns after antigen stimulation. (C) 1996 Academic Press, Inc.