Antibody treatment of LFA-1 on Jβ2.7 cells or T blasts leads to changes in integrin β₁-phosphorylation, activation epitope, and protein complexes. (A) Jβ2.7/LFA-1 cells or T blasts were treated with SDF1-α or antibodies to LFA-1. Cells were lysed and analyzed by western blotting for β₁/pThr-788/789 or β₁. (B) Cells were preincubated with Tiam1 inhibitor or (C) cytochalasin D (0.1 μg/mL) (C) and LFA-1 antibodies CBR LFA-1/2 or CBR LFA-1/7, and lysates analyzed as earlier. (D) T blasts were treated as earlier, allowed to adhere on poly-l-lysine, and fixed before immunofluorescence staining with an α₄ antibody or the antibody for activated integrin β₁ (12G10-488). α₄β₁ clustered by soluble VCAM-1 and VCAM-1 antibody. (E) Positive pixels from 50 cells stained with 12G10-488 were quantified using ImageJ. Standard deviations shown and statistical significance compared with control. *P < .05; **P < .01. Scale bar represents 10 μM. (F) Jβ2.7/LFA-1 cells or T blasts were lysed, immunoprecipitated with the activating CBR LFA-1/2 (1/2) or neutral TS2/4 (2/4) antibody, and immunoblotted for β₂, talin or 14-3-3. The unbound fraction of the CBR LFA-1/2 (ub1/2) and TS2/4 (ub2/4) precipitates were further immunoprecipitated with α₄ antibody and immunoblotted for α₄, talin, and 14-3-3.