DC-SIGN is present within lipid rafts in MDDCs and coprecipitates with Lyn and Syk tyrosine kinases. (A-B) Immature MDDCs from 2 independent donors were lysed in 1% Brij 98 lysis buffer at 37°C and fractionated by sucrose density gradient centrifugation as described in “Materials and methods.”¹⁴  The low-density Brij 98–insoluble fractions 2 to 4 (lanes 2-4, rafts) and the high-density Brij 98–soluble fractions 6 to 8 (lanes 6-8, soluble) were separated by 12.5% SDS-PAGE under nonreducing conditions, and the distribution of DC-SIGN was determined by immunoblotting. Cytoskeletal-associated rafts (CARs), obtained by solubilization of the cell pellet with Brij 98 + octyl d-glucoside in lysis buffer, were analyzed in parallel (lane 9). (A) The left panel was intentionally exposed for longer than the blot section shown in the right panel. (B) The distribution of Syk, Lyn, ERK, Ras, and ganglioside GM1 in the distinct fractions was determined by immunoblotting with specific antibodies or cholera toxin–HRP (for GM1). (C) Coprecipitation of DC-SIGN, Lyn, and Syk tyrosine kinases. DC-SIGN was immunoprecipitated with the MR-1 antibody from lipid raft–containing fractions 2 to 4 (rafts), fractions 5 to 6 (intrm. indicates intermediate between the rafts and the soluble material), and the 7 to 8 soluble fractions (soluble), and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibodies specific for DC-SIGN, Lyn, Syk, and phosphotyrosine-containing proteins. As a control, an aliquot from the whole lysate before fractionation (WL indicates whole lysate) was analyzed in parallel. (D) Presence of DC-SIGN and Syk in Lyn immunoprecipitates. Lyn was immunoprecipitated (IP(Lyn)) from the lipid raft–containing fraction pool (rafts) or the soluble material–containing fraction pool (soluble), and the precipitated material was subjected to SDS-PAGE and immunoblotting with antibodies specific for Lyn, DC-SIGN, and Syk. As a control, aliquots from the rafts and soluble fraction pools before immunoprecipitation were analyzed in parallel. (E) Presence of DC-SIGN in the post–anti-Lyn immunoprecipitation flow-through. Post–anti-Lyn immunoprecipitation flow-through from either lipid rafts or soluble membrane fraction pools (those indicated in D) were subjected to a further immunoprecipitation with a monoclonal antibody against DC-SIGN (IP(MR1)), and the precipitated material was subjected to SDS-PAGE and immunoblotting with a polyclonal antibody against DC-SIGN (DSG1). As a control, aliquots from the rafts and soluble fraction pools before immunoprecipitation were analyzed in parallel.