Anti dap12

mAbs to human LSECtin were established by immunization of Balb/C mice with recombinant LSECtin extracellular domain protein. Three independent clones, CCA023 (IgG2a), CFD051 (IgG1) and CCB059 (IgG2b), were established [34 (link)]. The anti-human LSECtin mAb CCB059 (IgG2b) was selected for staining by flow cytometry. The mouse IgG1 isotype control was from R&D Systems (Minneapolis, MN, USA). mAbs against human HLA-DR, CD83 and CD86 were from eBioscience (San Diego, CA, USA); mAbs against human CD40 and CD80 were from Biolegend (San Diego, CA, USA); anti-phosphotyrosine Ab (4G10) was from Millipore; anti-DAP12 and the other phospho-specific Abs were from Cell Signaling Technology (Danvers, MA). The Syk inhibitors piceatannol and R406 were purchased from Calbiochem (San Diego, CA, USA) and Selleckchem (Houston, TX, USA) respectively. Raf-1 inhibitor GW5074 was purchased from Calbiochem (San Diego, CA, USA); The MyD88 inhibitory peptide Pepinh-MYD was from InvivoGen (San Diego, CA, USA). The GlcNAc β1-2Man disaccharide was purchased from Dextra Laboratories (Reading, UK).

Total lysate from different cell lines were obtained using lysis buffer [50 mM Tris‐Cl (pH 8.0), 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulphonyl fluoride, 0.5 μg/ml leupeptin, and 1 μg/ml aprotinin]. For each sample, 40 μg of proteins were loaded and separated onto gradient SDS‐polyacrylamide gel (Bio‐Rad, Marnes‐La‐Coquette, France) for 1 hr 30 at 120 V, then transferred on PVDF membrane (Bio‐Rad). After a blocking step (nonfat milk), membranes were incubated overnight at 4°C with primary antibodies diluted in blocking solution (anti‐CHI3L1 from Abcam, Paris, France; anti‐PRUNE2 and anti KLRC3 from Abnova, Paris, France, anti‐DAP12 from Cell Signaling, Paris, France), followed by incubation with the appropriate secondary HRP‐conjugated antibodies. Blots were developed with Immobilon Western chemiluminescent HRP substrate (Millipore Fontenay‐Sous‐Bois, France) and analysed with G‐Box (Ozyme; Fisher Scientific, Illkirch, France). Band intensities were determined by densitometry using ImageJ software (NIH, Bethesda, MD, USA).