Fig. 4 mAb-independent and mAb-induced Internalization of PSMA

Figure 4. mAb-independent (A,C) and mAb-induced (B,D) internalization of PSMA. Cell surface was biotinylated at 40C with NHS-SS-biotin and then transferred to 370C. Biotin was reduced at 40C with glutathione as described in Materials and Methods. PSMA was immunoprecipitated, run on a 10% SDS-PAGE and detected with 125I-streptavidin. Lane 1, incubation at 40C, no reduction (100% control). Lane 2, incubation at 40C, followed by reduction (0% control). Lanes 3-6, incubation at 370C for 20, 60, 120 and 240 min followed by reduction. Note compete reduction of biotinylated PSMA in lane 2 compared to lanes 3-6 indicating that PSMA is internalized. Densitometric quantitation (C) revealed that approximately 60% of the total cell surface PSMA is internalized. Monoclonal antibody J591 induced internalization of PSMA (B,D). LNCaP cells biotinylated at 40C and incubated with different amounts of mAb J591 for 20 min at 370C. Lane 1, incubation at 40C, no reduction. Lane 2, incubation of 40C, reduction. Lanes 3-7, incubation at 370C with 0, 1, 2, 4 and 8 (g/ml of J591, reduction. Note the increased uptake of PSMA in the presence of 1 (g/ml of J591 (Lane 4) compared to Lane 3 where no antibody is present. Increasing mAb J591 concentration above 1 (g/ml did not increase the uptake indicating a saturation of PSMA uptake. Densitometric quantitation (D) revealed that 1 (g/ml of mAb J591 increased the uptake of PSMA by approximately 3-fold.