Function of Rac and Rho in CD99 signaling pathway. (A) Blocking of CD99 MoAb-induced aggregation of IM9 cells by C3 exoenzyme or L61F37A Rac mutant. Normal IM9 cells pretreated with (b) or without (a) the inhibitor of C3 transferase (20 μg/mL; UBI, Lake Placid, NY) for 36 hours were dispersed into single cells by repeated pipetting, incubated with control antibody or anti-CD99 MoAb for 1 hour, and assayed for cell aggregation. L61-TF (c) or L61F37A-TF (d) IM9 transfectants were stimulated with anti-CD99 MoAb for 1 hour and assayed for aggregation. (B) Gene constructs that contain L61 or L61F37A Rac in sense orientation and CD99 in antisense orientation. (C) Immunophenotypic analysis. All transfectants were examined for CD99 expression by flow cytometric analysis. L61 Rac rescues cell morphology from the H-RS phenotype, with a concomitant decrease in forward and side scatter and increased MHC class I expression, in CD99-deficient IM9 cells.