Downregulation of GNB isoforms or GNAI2 strongly affects LFA-1 function. HL60 cells transduced with a nonsilencing scrambled (scr) construct or with shRNAs against GNB1, GNB2, GNB4, GNB5, or GNAI2 were used. (A) Number of adherent cells per field of view (FOV) in the adhesion flow chamber. Chambers were coated with P-selectin, IL-8, and either a control IgG antibody or with the reporter mAb24 recognizing high-affinity LFA-1 (n = 4 experiments). *P < .05 vs all other groups. (B) Transduced HL60 cells were left untreated or were stimulated with IL-8, fixed with paraformaldehyde (4%), and stained with anti-CD11a and AF488-labeled secondary antibody to visualize clustered LFA-1 (n = 3 experiments). *P < .05 vs all other groups. (C) Flow cytometry analyzed allophycocyanin (APC)-labeled ICAM-1 binding in unstimulated (unst.) or IL-8 (100 ng/mL) stimulated cells (n = 4 experiments). *P < .05 vs all other groups. (D) Cells were left unstimulated or were stimulated with IL-8 (100 ng/mL, 3 minutes). The reaction was stopped by adding trichloroacetic acid, which was later removed by adding 1,1,2-trichloro-trifluoroethane-trioctylamine. The aqueous IP₃-containing supernatant was used for a competitive radioreceptor assay. Bars indicate IP₃ concentration in picomoles per 1 × 10⁷ HL60 cells (n = 3 experiments). *P < .05. (E) Concentration of intracellular calcium measured in Indo-1-labeled HL60 cells before and after chemokine stimulation. Arrow indicates IL-8 or LTB₄ stimulation (n = 4 experiments). #P < .05 vs all other groups. MFI, mean fluorescent intensity.