Figure 5
Figure 5. DC-SIGN colocalized with C1q and gC1qR on DCs. (A) DC-SIGN was cocaptured with C1q from iDC lysates. Whole-cell lysates (day 3) were added to microtiter plates coated with mAb DC-SIGN and the presence of C1q was detected using anti-C1q pAb. One representative experiment is shown (n = 3). (B) DC-SIGN colocalizes with C1q on the surface of iDCs in vitro, on DC precursors in blood and on iDCs in human tonsils in vivo. PBMCs, day 3 iDCs, and cryostat tonsil sections were analyzed for DC-SIGN and C1q. Cells and tonsil sections were incubated with rat anti–DC-SIGN and goat anti-C1q Abs or isotype controls, followed by FITC–anti–rat and PE-anti–goat secondary Abs and DAPI (blue). The slides were viewed using a Zeiss Axiovert 200M digital deconvolution microscope (63×; oil) and analyzed with AxioVision Version 4.8 software. Isotype controls showed little or no staining (data not shown; n = 6). (C) DC-SIGN binds to gC1qR. Antigen-capture ELISA experiments were performed using purified, soluble gC1qR. gC1qR was added to DC-SIGN–coated plates and binding was detected using anti-gC1qR pAb. One representative experiment is shown (n = 3). (D) C1q increased the binding of gC1qR to DC-SIGN significantly. ELISA experiments were performed using 5 μg/mL of biotinylated gC1qR premixed with increasing concentrations (0-50 μg/mL) of C1q. Biot-gC1qR/C1q was added to DC-SIGN– or 5% nonfat milk–coated plates and binding was detected using neutravidin-AP. One representative experiment is shown (n = 2). *P < .05. (E) DC-SIGN was cocaptured with gC1qR from iDC lysates. Antigen-capture ELISA experiments were performed using whole-cell DC lysates (day 3). DC lysates were added to microtiter plates coated with rat anti–human DC-SIGN and the presence of gC1qR was detected using anti-gC1qR pAb. One representative experiment is shown (n = 3). (F) DC-SIGN colocalizes with gC1qR on the surface of blood DC precursors and iDCs. PBMCs and day 3 iDCs were incubated with rat anti–DC-SIGN and rabbit anti-gC1qR Abs or isotype controls, followed by FITC-anti–rat and PE–anti–rabbit secondary Abs and DAPI (blue). The slides were viewed using a Zeiss Axiovert 200M digital deconvolution microscope (68×; oil) and analyzed with AxioVision Version 4.8 software. Isotype controls showed little or no staining (data not shown; n = 7).

DC-SIGN colocalized with C1q and gC1qR on DCs. (A) DC-SIGN was cocaptured with C1q from iDC lysates. Whole-cell lysates (day 3) were added to microtiter plates coated with mAb DC-SIGN and the presence of C1q was detected using anti-C1q pAb. One representative experiment is shown (n = 3). (B) DC-SIGN colocalizes with C1q on the surface of iDCs in vitro, on DC precursors in blood and on iDCs in human tonsils in vivo. PBMCs, day 3 iDCs, and cryostat tonsil sections were analyzed for DC-SIGN and C1q. Cells and tonsil sections were incubated with rat anti–DC-SIGN and goat anti-C1q Abs or isotype controls, followed by FITC–anti–rat and PE-anti–goat secondary Abs and DAPI (blue). The slides were viewed using a Zeiss Axiovert 200M digital deconvolution microscope (63×; oil) and analyzed with AxioVision Version 4.8 software. Isotype controls showed little or no staining (data not shown; n = 6). (C) DC-SIGN binds to gC1qR. Antigen-capture ELISA experiments were performed using purified, soluble gC1qR. gC1qR was added to DC-SIGN–coated plates and binding was detected using anti-gC1qR pAb. One representative experiment is shown (n = 3). (D) C1q increased the binding of gC1qR to DC-SIGN significantly. ELISA experiments were performed using 5 μg/mL of biotinylated gC1qR premixed with increasing concentrations (0-50 μg/mL) of C1q. Biot-gC1qR/C1q was added to DC-SIGN– or 5% nonfat milk–coated plates and binding was detected using neutravidin-AP. One representative experiment is shown (n = 2). *P < .05. (E) DC-SIGN was cocaptured with gC1qR from iDC lysates. Antigen-capture ELISA experiments were performed using whole-cell DC lysates (day 3). DC lysates were added to microtiter plates coated with rat anti–human DC-SIGN and the presence of gC1qR was detected using anti-gC1qR pAb. One representative experiment is shown (n = 3). (F) DC-SIGN colocalizes with gC1qR on the surface of blood DC precursors and iDCs. PBMCs and day 3 iDCs were incubated with rat anti–DC-SIGN and rabbit anti-gC1qR Abs or isotype controls, followed by FITC-anti–rat and PE–anti–rabbit secondary Abs and DAPI (blue). The slides were viewed using a Zeiss Axiovert 200M digital deconvolution microscope (68×; oil) and analyzed with AxioVision Version 4.8 software. Isotype controls showed little or no staining (data not shown; n = 7).

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