Fig. 3. Expression of a CD44 variant extends the ligand binding capacity of the lymphoma cells. (A) Western blots of CPC-precipitated CD44 derived from a total cell population of LB cells transfected with CD44v4-v10 cDNA (LB-TRv) or standard CD44 (LB-TRs). LB-TRv and LB-TRs cells were lysed and mixed with KS, HA, CS, H (Heparin) and HS. After CPC precipitation of GAG-associated proteins and their resolution on 7.5% SDS-PAGE (under nonreducing conditions), they were identified by western blot with KM81 anti-CD44 mAb. The last lane in each panel (marked by C) shows control precipitation (i.e. without the prior addition of GAGs). Lanes marked by ‘lysate’ show western blotting of the total cell lysate (no CPC precipitation) with anti-CD44 mAb. CD44v of LB-TRv cells binds HA as well as additional GAGs. (B) Flow cytometry analysis of CS binding to total LB-TRv and LB-TRs cell populations. LB-TRv and LB-TRs cells were stained with fluoresceinated CS (50 µg/ml) in the absence (FL-CS) or presence of KM81 anti-CD44 mAb (100 µg/ml) (FL-CS+KM81). Separate histograms display CS binding; matched histograms show the inability to bind CS. AF, autofluorescence.