293 cells were transfected with siRNA targeting human Cbl-b or with control nontargeting siRNA. After 24 hours, the cells were transiently transfected with either WT human LFA-1 or mutant human T758A β2 LFA-1. The expression of WT and mutant LFA-1 were comparable and not affected by Cbl-b knockdown (Figure S5). (A) Cbl-b knockdown was verified by Western blot using an antibody to human Cbl-b. As a control, Cbl-b knockdown did not affect the expression of β-actin or of the transfected β2-integrin (CD18). (B) 293 cells were transfected with control siRNA or siRNA targeting Cbl-b and then with WT or mutant LFA-1 (αL DNA combined with WT β2 or T758A β2) or mock transfected as control, and were allowed to adhere to immobilized ICAM-1. Adhesion is shown in the absence (□) or presence (■) of PMA (50 ng/mL) or LFA-1–activating mAb, MEM-83 (10 μg/mL; ▩). Cell adhesion is represented as the percentage of adherent cells. Data are means plus or minus SD. *P < .05; ns, nonsignificant.