American Society of Hematology

Figure 4.

$Figure 4. DC-SIGN is present within lipid rafts in lymphoid cells and coprecipitates with tyrosine kinases. (A) Jurkat-DC-SIGN (left panels) and mock-transfected Jurkat cells (right panels) were lysed in 1% Brij 98 lysis buffer at 37°C, and Brij 98–insoluble fractions 2 to 4 (lanes 2-4, rafts) and the high-density Brij 98–soluble fractions 7 to 8 (lanes 7-8, soluble) were separated by 12.5% SDS-PAGE under nonreducing conditions. 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). The distribution of DC-SIGN, Lck, ZAP-70, LAT, CD3ϵ, and ganglioside GM1 in the distinct fractions was determined by immunoblotting with specific antibodies or cholera toxin–HRP (for GM1). (B) Coprecipitation of DC-SIGN and tyrosine kinases. DC-SIGN was immunoprecipitated with the MR-1 antibody (MR1 ip) from lipid raft–containing fractions 2 to 4 (rafts, lane 2), fractions 5 to 6 (between the rafts and the soluble material, lane 3), and the 7 to 8 soluble fractions (sol., lane 4), and the immunoprecipitated material was subjected to SDS-PAGE and immunoblotting with antibodies against DC-SIGN, Lck, or ZAP-70. As a control, fractions containing either rafts (lane 1) or cytoskeletal-associated rafts (CARs, lane 5) were analyzed in parallel. (C) Jurkat-DC-SIGN cells were cell-surface labeled with biotin, lysed in 1% Brij 98 lysis buffer at 37°C, and Brij 98–insoluble fractions (lanes 1, rafts), intermediate fractions (lanes 2), high-density Brij 98–soluble fractions (lanes 3, sol.), and cytoskeletal-associated raft-containing fractions (CARs, lane 4) were obtained. An aliquot from each fraction was removed and analyzed by 12.5% SDS-PAGE under nonreducing conditions and subjected to Western blot with anti–DC-SIGN or anti-LAT polyclonal antisera (left panel). Then, fractions were subjected to pull-down with Streptavidin-agarose, and the immunoprecipitated material was separated by 12.5% SDS-PAGE under nonreducing conditions and subjected to Western blot with anti–DC-SIGN or anti-LAT polyclonal antisera (right panel). Each experiment was performed twice with similar results, and one of the experiments is shown.$

DC-SIGN is present within lipid rafts in lymphoid cells and coprecipitates with tyrosine kinases. (A) Jurkat-DC-SIGN (left panels) and mock-transfected Jurkat cells (right panels) were lysed in 1% Brij 98 lysis buffer at 37°C, and Brij 98–insoluble fractions 2 to 4 (lanes 2-4, rafts) and the high-density Brij 98–soluble fractions 7 to 8 (lanes 7-8, soluble) were separated by 12.5% SDS-PAGE under nonreducing conditions. 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). The distribution of DC-SIGN, Lck, ZAP-70, LAT, CD3ϵ, and ganglioside GM1 in the distinct fractions was determined by immunoblotting with specific antibodies or cholera toxin–HRP (for GM1). (B) Coprecipitation of DC-SIGN and tyrosine kinases. DC-SIGN was immunoprecipitated with the MR-1 antibody (MR1 ip) from lipid raft–containing fractions 2 to 4 (rafts, lane 2), fractions 5 to 6 (between the rafts and the soluble material, lane 3), and the 7 to 8 soluble fractions (sol., lane 4), and the immunoprecipitated material was subjected to SDS-PAGE and immunoblotting with antibodies against DC-SIGN, Lck, or ZAP-70. As a control, fractions containing either rafts (lane 1) or cytoskeletal-associated rafts (CARs, lane 5) were analyzed in parallel. (C) Jurkat-DC-SIGN cells were cell-surface labeled with biotin, lysed in 1% Brij 98 lysis buffer at 37°C, and Brij 98–insoluble fractions (lanes 1, rafts), intermediate fractions (lanes 2), high-density Brij 98–soluble fractions (lanes 3, sol.), and cytoskeletal-associated raft-containing fractions (CARs, lane 4) were obtained. An aliquot from each fraction was removed and analyzed by 12.5% SDS-PAGE under nonreducing conditions and subjected to Western blot with anti–DC-SIGN or anti-LAT polyclonal antisera (left panel). Then, fractions were subjected to pull-down with Streptavidin-agarose, and the immunoprecipitated material was separated by 12.5% SDS-PAGE under nonreducing conditions and subjected to Western blot with anti–DC-SIGN or anti-LAT polyclonal antisera (right panel). Each experiment was performed twice with similar results, and one of the experiments is shown.

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