RANTES specifically binds to cell surface-expressed GAGs. (A) HeLa-CD4 cells were treated (+) for 24 hours with 640 nM RANTES or were mock-treated (–). Cell extracts were prepared and immunoprecipitated with specific antibodies as indicated. Samples were separated on 10% SDS-PAGE gels and Western blots were developed using polyclonal anti-RANTES antibodies. When indicated, GAG-digestion (+) with heparinase/chondroitinase-ABC enzymes was performed after cell lysis and IP. C indicates control, 5 μg RANTES directly loaded for gel analysis; HS, heparan sulfate; CS, chondroitin sulfate; Rd, RANTES dimer. (B) HeLa-CD4 cells were treated as described in panel A with the exception that GAG digestion was performed after RANTES treatment and prior to IP. (C) Primary PHA-activated CD4⁺ T cells were incubated for 24 hours with 640 nM RANTES or mutants Met-RANTES, Met-RANTES-E66S, or [⁴⁴AANA⁴⁷] RANTES. Binding of RANTES or derivatives to cells was monitored by IP and immunoblotting with polyclonal anti-RANTES preparation. (D-E) Binding of RANTES (50 nM or 640 nM) and [⁴⁴AANA⁴⁷] RANTES (640 nM) to HeLa-CD4 cells (D) or primary PHA-activated CD4⁺ T cells (E) after 24 hours of incubation with the chemokines was monitored by FACS analysis using PE-labeled anti-RANTES antibodies. One of 2 to 4 individual experiments is shown.