Fig. 3

Immunoprecipitation of ICAM-1 from confluent T84 monolayers following selective cell surface labeling (apical or basolateral biotinylation)

T84 monolayers cultured to confluency on 5-cm² permeable supports were cooled to 4°C and selectively labeled on the apical (Lanes A) or basolateral surface (Lanes B) with biotin followed by immunoprecipitation of ICAM-1 using mAb R6.5 as described in Materials and Methods. Immunoprecipitates were subjected to SDS-PAGE on 5–16% Polyacrylamide gradient gels followed by Western blotting, incubation with streptavidin-peroxidase, and development by enhanced chemiluminescence. The immunoprecipitates of control, unstimulated monolayers ([−] IFN) were compared with those of monolayers maximally stimulated with IFNγ ([+] IFN). As can be seen, there is no ICAM-1 in the apically (A) or basolaterally labeled (B) unstimulated monolayers ([−] IFN). However, following IFNγ stimulation (1000 U/ml, 48 hr) there is the appearance of a ∼100 kD ICAM-1 band in the apically labeled (A) but not the basolaterally labeled (B) lanes ([+] IFN). Ctl, control immunoprecipitation with normal mouse IgG; this lane shows a nonspecific, prominent 70-kD band which is also observed in the ICAM-1 lanes.