Fig 2: mRNA expression in EL4 cells stimulated with or without anti-CD3/CD28 antibodies or anti-CD3/CD28/OCILRP2 antibodies.RNA from un-stimulated or CD3/CD28 antibody- or CD3/CD28/OCILRP2 antibody-stimulated EL4 cells was isolated using the RNeasy kit (Qiagen). cDNA of IL-2, NF-κB, NFAT, MAPK3, and MAPK8 or the control household gene β-actin was amplified using SYBR Premix Ex Taq (RRO41A, TaKaRa, Shiga, Japan), and expression was monitored using an Rotor-gene 6000 real-time platform (Corbett Research, Australia). Ct values were normalized for the expression of the β-actin gene. *P<0.05.

Fig 3: IL-2 secreted from EL4 cells after stimulation with an anti-CD3/CD28 mAb or PMA/ionomycin.EL4 cells were incubated with or without anti-CD3/CD28 antibodies (at various concentrations) or PMA (10 ng/mL) and ionomycin (100 ng/mL) for 12, 24, and 48 h. Supernatants were obtained by centrifugation for 3 min using a microcentrifuge. Cytokine IL-2 secreted into the supernatant was determined by ELISA.

Fig 4: Western blot analysis of Raf, Erk, Jnk, and Akt phosphorylation in EL4 cells.EL4 T cells were incubated in DMEM medium with an immobilized anti-CD3/CD28 mAb in the presence or absence of si-OCILRP2. After incubation at 37°C for 48 h, the T cells were lysed, and aliquots of 20 µg of the whole-cell lysate were analyzed by western blotting using antibodies against phospho-Erk, Erk, phospho-Jnk, and Jnk (a) and phospho-Raf and phosphor-Akt (b). The data are expressed as a percentage of the level of active Erk or Jnk of the total Erk or Jnk. Each data point represents the mean ± S.E. from three separate experiments.

Fig 5: Membrane co-localization of OCILRP2 and DAP12 under anti-CD3/CD28 mAb treatment in EL4 T cells.EL4 T cells pre-transfected with pEGFP-siOCILRP2 or pre-coated with the OCILRP2 Ab were cultured overnight in the presence or absence of an anti-CD3/CD28 mAb and then stained for OCILRP2 (green) and nuclear stained with DAPI (blue) and DAP12 (red) to study OCILRP2 and DAP12 protein expression and localization. Unstimulated EL4 T cells exhibited OCILRP2 protein expression in the cytoplasm (upper panels). In contrast, CD3/CD28-activated EL4 T cells showed intracellular and membrane OCILRP2. OCILRP2/DAP12 co-localization appears in yellow. Each picture is representative of the vast majority of the observed cells on the slides.