FIGURE 2.

Verification of Siglec-5 expression by human T cells. (a) CD3+ T cells were enriched from adult PBMCs and cultured with plate bound anti-CD3 and anti-CD28 stimulation and IL-2, for up to 4 days. mRNA was prepared at each time point and Siglec-5 mRNA was detected using qPCR. (b) Representative blot (n = 3) of CD3+ T cells enriched from adult PBMCs cultured with plate bound anti-CD3 and anti-CD28 stimulation and IL-2, for up to 3 days. At each time point, cell lysates were prepared using denaturing non-reducing conditions. (c) and (d) Cord blood T cells were stimulated with soluble anti-CD3 and IL-2 for 2 days. Cells were lysed with mild detergent (0·5% NP-40) and proteins were immunoprecipitated (IP) using anti-Siglec-5/14 mAb (clone 1A5) or mIgG1 isotype CNBr conjugated beads. As controls, Jurkat T cells were transfected with Siglec-5, Siglec-14 or empty vector and whole cell lysates were prepared. Where indicated, IP-ed proteins were treated with PNGase F to remove N-linked glycosylation. All samples were run on SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with anti-Siglec5/14 polyclonal antibody. Representative membranes of 2 independent repeats