Mouse igg2a

B cells were isolated using CD19 Microbeads (Miltenyi Biotec) and cultured at 2.5×10⁶ cells/ml for 48h in the presence of soluble recombinant human CD40L (100ng/ml) (MegaCD40L, Enzo LifeSciences) or PBS as control before cells were lysed using ice cold IP Lysis Buffer (Pierce) in the presence of protease inhibitors. 500 µg protein lysate was used for each IP using the Immunoprecipitation Kit Dynabeads® Protein G (Life Technologies). Briefly, 5 µg mAb (CD45, clone F10–89-4) or isotype control (mouse IgG2a, BioRad) was bound to 50 µL Dynabeads and crosslinked using BS3 reagent (ThermoFisher) according to manufacturers instructions, before cell lysate was added at 4°C for 4h. Beads were washed and bound protein was eluted and sample buffer (NuPAGE LDS sample buffer) and DTT was added. Samples were heated to 70°C for 10 min before loaded onto SDS-PAGE. Gels were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked in 5% nonfat dry milk and 0.1% Tween. The membrane was probed with goat anti-Galectin-1 polyclonal antibody (1:2000)(R&D Systems) and anti-goat IgG HRP (1:1000)(R&D Systems) or anti-CD45 (1:1000) and anti-mouse IgG HRP (1:1000) (R&D Systems).