Af3370

The procedure for immunofluorescence (IF) was performed as previously described [16] (link). Briefly, EL4 cells transfected overnight with pEGFP-C3-siOCILRP2 were stimulated with anti-CD3/CD28 antibodies. For the other groups, EL4 cells were stimulated with or without anti-CD3/CD28 or anti-CD3/CD28/OCILRP2 antibodies. All cells were incubated on glass cover slips and fixed with 3.5% paraformaldehyde for 15 min, which was then stopped by the addition of 30 mM glycine. After washing, the cells were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 3% bovine serum albumin in PBS for at least 1 h (4°C). A polyclonal anti-OCILRP2 antibody (AF3370, R&D systems, USA) and monoclonal anti-DAP12 antibody (sc-133174, Santa Cruz, USA) were used at a 1∶2000 dilution. Goat anti-mouse-IgG-PE (sc-3738, Santa Cruz, USA) and rabbit anti-goat-IgG-FITC (sc-2777, Santa Cruz, USA) secondary antibodies were used at a 1∶100 dilution. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI) (D9542, Sigma-Aldrich, USA), and the cells were examined by confocal microscopy (98DDFR/470111CR, Bio-Rad, USA).

EL4 (ATCC TIB 181) cells were purchased from American Type Culture Collection and cultured as described [15] (link). The EL4 cells were stimulated for the indicated times with combinations of anti-CD3 (sc-18871, Santa Cruz, USA) and/or anti-CD28 antibodies (sc-12727, Santa Cruz, USA). In some experiments, an anti-IL-2 antibody (H-20, Santa Cruz, USA) or anti-OCILRP2 antibody (AF3370, R&D systems, USA) was added to the culture medium. Controls were stimulated with phorbol myristate acetate (PMA) (p1585, 50 ng/mL, Sigma, USA) and ionomycin (I3909, 100 ng/mL, Sigma, USA).