Mab1859

2B4 cells expressing hDectin-1A or 1B (2×10⁶ for glycosidase analysis, 1×10⁸ for OpeRATOR treatment) and splenocytes (6×10⁷) isolated from hDectin-1 transgenic mice were lysed with NP-40 lysis buffer. The solubilized hDectin-1 was captured on beads with an anti-hDectin-1 mAb (MAB1859, R&D Systems, Minneapolis, MN). For glycosidase analysis, the immobilized proteins were treated with PNGase F or PNGase F plus O-glycosidase mix including Neuramindase, Hexosaminidase, and O-glycosidase (New England Biolabs, Beverly, MA) according to the manufacturer’s instruction. O-glycosylated peptides were liberated by OpeRATOR and SialEXO (Genovis, Lund, Sweden) according to the manufacturer’s instructions, electrophoresed through 10% acrylamide (Nacalai Tesque) or 10–20% gradient gels (ATTO, Tokyo, Japan) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). PVDF membranes were detected using anti-hDectin-1 polyclonal antibodies (AF1859, R&D Systems), followed by incubation with an HRP-labeled secondary IgG (Invitrogen, Carlsbad, CA) or Protein G (Sigma-Aldrich, St. Louis, MO). Immunocomplexes were detected using Chemi-Lumi One Super (Nacalai Tesque) and a Luminescent Image Analyzer LAS-1000 (Fujifilm, Tokyo, Japan).

Human macrophages were differentiated at day 5, and activated with IL-4 (20 ng/ml) for additional 48 h. At day 7, the M(IL-4) macrophages were washed with warmed RPMI-1640 medium supplemented with M-CSF (10 ng/ml) and pre-incubated for 30 min with 10 µg/ml blocking antibodies specific to either Dectin-1 (MAB1859, R&D Systems) or DC-SIGN (ab13847, abcam), or both. As a control, M(IL-4) macrophages were incubated with an irrevelevant antibody (Ab-Control). For efficient stimulation of Dectin-1, M(IL-4) macrophages were treated with cytochalasin D (1 µg/ml, C8273, SIGMA) in combination with purified β-glucan from Saccharomyces cerevisiae (10 µg/ml, G5011, SIGMA), as previously described (31 (link)); for DC-SIGN, cells were stimulated with 10 µg/ml of ManLAM (kindly provided by Dr. Jerome Nigou, IPBS/CNRS); and for TLR-4, cells were stimulated with 1 µg/ml of LPS. After 24 h, the supernatants were collected and stored at −80°C until further use for ELISA analysis.