LFA-1 cytoplasmic regions regulate transient and stable arrest. (A) Schematic representation of the cytoplasmic region of human β2 subunit. Deletions and point mutation were made at the indicated amino acid residues. Deletion mutants include the indicated amino acids. The boxes indicate the NPXF/Y motif, which is the talin-binding region. (B) Under flow adhesion of BAF cells expressing L-selectin and LFA-1 mutants with or without CXCL12. Data represent the mean ± SD of 3 independent experiments. (C) Expression of activation epitopes detected by mAb KIM127 (left) and MEM148 (right) in unstimulated LFA-1 mutants. (D) Induction of the arrest by a penetratin-1 fusion peptide corresponding to β2/745-750. Treatment with peptide β2/745-750 (SQWNND), but not with the control peptide (SQANND; 100 μg/mL), induced the stable arrest of BAF/L-selectin/LFA-1 cells. The cells were treated with the peptides in the absence or presence of CXCL12 as described in the supplemental Methods. Data represent the mean ± SD of 3 independent experiments. *P < .01, compared with the cells with control peptide. (E) Knockdown of Rap1 in BAF cells/L-selectin expressing αL/β2 W747A. (Top panel) Western blotting of vector alone (ct) or vector encoding Rap1-specific shRNA (Rap1 KD). Tubulin served as a loading control. (Bottom panel) Under flow adhesion of BAF cells/L-selectin expressing αL/β2 W747A mutation infected with lentiviruses encoding GFP alone (ct) or Rap1a/b-specific shRNA (KD). Data represent the mean ± SD of 3 independent experiments. *P < .005, compared with control lymphocytes.